JP7636752B2 - Alkyne compounds, vitamin D compounds, analytical methods, and production methods - Google Patents

Description

The present invention relates to an alkyne compound and a vitamin D compound. The present invention also relates to an analytical method using the vitamin D compound and a method for producing the vitamin D compound.

There is a high demand in the clinical and food nutrition fields for the detection and quantification of vitamin D compounds in living organisms. For example, detection and quantification of vitamin D metabolites in biological samples such as blood, urine, body fluids, and cerebrospinal fluid have been performed. Several techniques for the analysis of vitamin D compounds have been proposed.

For example, Non-Patent Document 1 below describes the synthesis of deuterated analogs of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Also, Non-Patent Document 2 below describes the differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling.

Rahul Ray et al., Synthesis of 25-hydroxy-[6,19,19'-2H3vitamin D3 and 1α,25-dihydroxy-[6,19'-2H3]vitamin D3, Steroids, 1992, 57, 142-146. Martin Kaufmann et al., Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling, J. Bone Miner. Res., 2021, 36, 1340-1350. Aileen Mendoza et al., Controlled lipid β-oxidation and carnitine biosynthesis by a vitamin D metabolite, Cell Chem. Biol., 28, Published: September 09, 2021 Masahiko Seki et al., A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses, Rapid Commun. Mass Spectrom., 2020, 34, e8648.

The present invention aims to provide compounds that are useful for the analysis of vitamin D compounds.

The present inventors have found that a specific vitamin D compound labeled with a stable isotope is useful for the analysis of vitamin D metabolites. The present inventors have also found that the vitamin D compound can be produced by a specific alkyne compound.

That is, the present invention provides the following.
[1] An alkyne compound represented by the following formula (100):
[Chemical formula 1-1]
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
[Chemical formula 1-2]
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
(However, the following alkyne compounds:
[Chemical formula 1-3]
(Excluding.)
[2] An alkyne compound represented by the following formula (100):
[Chemical formula 2-1]
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
wherein at least one of _X21 , _X22 , _X23 , and _X24 is any one of CHY, CDY, ^13CHY , and ^13CDY , where Y is any one of _{NH2, 15NH2 ,} OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and ^a sugar substituent;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
[Chemical formula 2-2]
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
[3] The alkyne compound according to the above [2], wherein in the formula (100), at least one of X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ is CHY or CDY.
[4] A vitamin D compound represented by the following formula (200):
[Chemical formula 3-1]
[In formula (200),
A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound;
X' ₁ is C or ¹³ C;
X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ each independently represent any one selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y' is any one _of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other;
R' is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X'3 is C or ^13C ;
At least one of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N;
P is a _C1 - _C10 alkyl group, which may or may not be substituted with a hydroxy group, or a _C2 - _C10 alkenyl group, which may or may not be substituted with a hydroxy group, and the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group;
The sugar substituent has the structure:
[Chemical formula 3-2]
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
(However, the following vitamin D compounds:
[Chemical formula 3-3]
(Excluding.)
[5] A vitamin D compound represented by the following formula (200):
[Chemical formula 4-1]
[In formula (200),
A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound;
X' ₁ is C or ¹³ C;
X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ each independently represent any one selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y' is any one _of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other;
R' is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X'3 is C or ^13C ;
At least one of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N, wherein at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is modified with a stable isotope ¹³ C, ¹⁸ O or ¹⁵ N, or at least two of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ are modified with a stable isotope D, or at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is CDD;
P is a _C1 - _C10 alkyl group, which may or may not be substituted with a hydroxy group, or a _C2 - _C10 alkenyl group, which may or may not be substituted with a hydroxy group, and the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group;
The sugar substituent has the structure:
[Chemical formula 4-2]
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
[6] The vitamin D compound according to the above [3], wherein in formula (200), P has any one of the following structures:
[Chemical formula 5]
[In the structure relating to P,
Q, Q', Q'', Q''', and Q'''' each independently represent any of H, CH ₂ , CH ₃ , C ₂ H ₅ , O, OH, and a sugar substituent.
[7] An analytical method using the vitamin D compound according to [4] or [5] above.
[8] The method of analysis includes analyzing the sample by mass spectrometry;
In the analysis, the vitamin D compound is used as an internal standard.
The analysis method according to [7] above.
[9] The analysis method according to [8], wherein the mass spectrometry is LC-MS/MS or LC/MS.
[10] The analytical method according to [7], wherein a vitamin D compound not having a hydroxy group at position 1 of the A ring is analyzed separately from a vitamin D compound having a hydroxy group at position 1 of the A ring.
[11] The analysis method according to [10], wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 2-hydroxyvitamin D compound and a 4-hydroxyvitamin D compound.
[12] The method for analysis according to [10], wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 4β,25-dihydroxyvitamin D3 compound and a 4-hydroxyvitamin D compound.
[13] A method for producing a vitamin D compound according to the above [4] or [5], wherein an alkyne compound represented by the following formula (100) is used as a material for forming the A ring of the vitamin D compound.
[Chemical formula 6-1]
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
wherein at least one of _X21 , _X22 , _X23 , and _X24 is any one of CHY, CDY, ^13CHY , and ^13CDY , where Y is any one of _{NH2, 15NH2 ,} OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and ^a sugar substituent;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
[Chemical formula 6-2]
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]

By using the vitamin D compound according to the present invention, it is possible to detect and/or quantify vitamin D metabolites, and the vitamin D compound can be produced by using the alkyne compound according to the present invention.
The effects of the present invention are not limited to those described herein, and may be any of the effects described in this specification.

FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. FIG. 1 shows NMR spectrum data. This is an SRM chromatogram of a 25OHD ₃ -26R,23S-lactone-d ₃ preparation. This is an SRM chromatogram of a 1α,25(OH) ₂ D ₃ -26R,23S-lactone-d ₃ preparation. 1 shows a calibration curve for the 25OHD ₃ -26R,23S-lactone standard. This is a calibration curve for 1α,25(OH) ₂ D ₃ -26R,23S-lactone standard. This is an SRM chromatogram of 25OHD ₃ -26R,23S-lactone in pooled serum. SRM chromatogram of 1α,25(OH) ₂ D ₃ -26R,23S-lactone in pooled serum. FIG. 2 shows a reaction scheme for derivatization. FIG. 2 shows a reaction scheme for derivatization. This is a calibration curve for ₃ types of 25OHD. This is a calibration curve _for 24R,25(OH) _2D3 standard. FIG. ₁₃ is an SRM chromatogram of 25OHD3 in pooled serum. SRM chromatogram of 24R,25(OH) _2D3 in _pooled serum. The SRM chromatogram of the antibody column non-adsorbed fraction is shown. The SRM chromatogram of the antibody column adsorption fraction is shown. The calibration curve for 4β,25-(OH) ₂ D ₃ standard is shown. 1 shows an SRM chromatogram of the antibody column non-adsorbed fraction of pooled serum. 1 shows an SRM chromatogram of the antibody column adsorption fraction of pooled serum. FIG. 1 is a diagram illustrating derivatization of vitamin D compounds and cleavage of the derivatives. FIG. 1 shows the chemical structures of vitamin D compounds having a hydroxy group at position 1 of the A ring and vitamin D compounds not having a hydroxy group at position 1 of the A ring. FIG. 1 shows the chemical structures of 1α,25-dihydroxyvitamin _D3 and 4β,25-dihydroxyvitamin _D3 . An example of a chemical reaction scheme for producing the vitamin D compound of the present invention is shown below. An example of a chemical reaction scheme for producing the vitamin D compound of the present invention is shown below. FIG. 1 shows examples of Vitamin D compounds. FIG. 1 shows examples of compounds used to produce the alkyne compound of the present invention.

The following describes preferred embodiments of the present invention. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention.
The present invention will be described in the following order.
1. Description of the Invention 2. First embodiment (alkyne compound)
2-1. Structure of the alkyne compound of formula (I) 2-2. Structure of the alkyne compound of formula (100) 2-3. Method for using the alkyne compound 2-4. Method for producing the alkyne compound 3. Second embodiment (vitamin D compound)
3-1. Structure of vitamin D compound of formula (III) 3-2. Structure of vitamin D compound of formula (200) 3-3. Method of using vitamin D compound 3-4. Method of producing vitamin D compound 4. Third embodiment (analysis method using vitamin D compound)
4-1. Example of analytical method 4-2. Modified analytical method 5. Fourth embodiment (method of producing vitamin D compound)
6. Examples 6-1. Example 1: Production of alkyne compounds and vitamin D compounds 6-2. Example 2: LC-MS/MS analysis of _25OHD3-26R ,23S-lactone and _1α ,25(OH) _2D3-26R ,23S-lactone in pooled serum 6-3. Example 3: LC-MS/MS analysis _{of 25OHD3} and 24R,25(OH) _2D3 in pooled serum 6-4. Example 4: Separation and measurement _of 4β,25-(OH) _2D3 6-5. Example 5: LC-MS/MS analysis of _{4β,25-(OH)2D3 in} pooled serum

1. Description of the invention

Various vitamin D metabolites have been discovered so far. For example, one of the most important vitamin D metabolites in terms of physiological activity is 1α,25(OH) ₂ D ₃ , which is also called active vitamin D. Another example of vitamin D metabolite is vitamin D lactone, which has been reported to inhibit the metabolism of fatty acids (Non-Patent Document 3 above). Fine structural changes in the side chain structure of vitamin D metabolites can affect the physiological activity of vitamin D metabolites. To clarify the various physiological activities of each vitamin D metabolite, it is necessary to accurately quantify each vitamin D metabolite.

At present, 1α,25(OH) ₂ D ₃ is quantified by immunochemical measurement, but it is difficult to individually detect or quantify other trace vitamin D metabolites by immunochemical measurement. LC-MS/MS method using a derivatization reagent is expected as a method for detecting or quantifying vitamin D metabolites with high sensitivity (Non-Patent Document 4 mentioned above). For detection or quantification by LC-MS/MS method, vitamin D metabolites labeled with stable isotopes are essential.

As described in the above-mentioned non-patent document 1, a technique for deuterium labeling by H-D exchange using heavy water is known. However, while vitamin D compounds labeled by this method are stored in a solution, H-D exchange with light water in the solvent may occur. In other words, vitamin D compounds labeled with deuterium by H-D exchange have storage stability problems.

Vitamin D compounds have an A ring, a seco-B ring, a C ring, and a D ring (herein, the C ring and the D ring are collectively referred to as "CD ring"). The CD ring has different side chains depending on the type of vitamin D compound, i.e., it has structural diversity. The A ring portion of vitamin D compounds also has structural diversity, but the structural diversity is less than that of the CD ring side chain.
The present invention provides an alkyne compound labeled with a stable isotope. The alkyne compound can be used as a substance for forming the A ring portion. The vitamin D compound produced using the alkyne compound has its A ring portion labeled with a stable isotope. Since a stable isotope is introduced into the A ring portion by using the alkyne compound, various vitamin D metabolites labeled with a stable isotope can be produced without introducing a stable isotope into the CD ring side chain. Here, as described above, the structural diversity of the A ring portion of the vitamin D compound is smaller than that of the CD ring side chain. In other words, various vitamin D compounds can be produced by using fewer variations of the alkyne compound. Therefore, various vitamin D compounds labeled with a stable isotope can be efficiently produced by using the alkyne compound according to the present invention.

The vitamin D compound according to the present invention can be produced using the alkyne compound according to the present invention as a material for forming the A ring. The vitamin D compound does not need to be a compound labeled by H-D exchange using heavy water, and as a result, it has excellent storage stability.

The vitamin D compounds according to the present invention can be used in various analytical methods, particularly mass spectrometry, more particularly isotope dilution mass spectrometry. That is, the present invention also provides an analytical method using the vitamin D compounds. In one embodiment, the present invention provides an analytical method comprising detecting or quantifying a vitamin D metabolite. The detection or quantification may be performed by a mass spectrometer, particularly a liquid chromatography mass spectrometer, and even more particularly an LC-MS/MS. The vitamin D compounds according to the present invention can be used regardless of the manufacturer of the mass spectrometer.

Furthermore, the vitamin D compound according to the present invention can be used, for example, in the field of clinical testing or food testing. There is a very large demand for measuring vitamin D metabolites in the field of clinical testing or food testing. It is believed that the present invention can efficiently produce stable isotope-labeled compounds not only for currently known vitamin D metabolites, but also for vitamin D metabolites that may be discovered in the future. Therefore, the present invention also contributes to the rapid construction of a measurement environment for various vitamin D metabolites.

The present invention is described in more detail below.

2. First embodiment (alkyne compound)

The present invention provides an alkyne compound represented by the following formula (I). The alkyne compound is labeled with a stable isotope as described below. The alkyne compound can be used to produce various vitamin D compounds, and more specifically, the alkyne compound may be used as a compound that forms the A ring portion of various vitamin D compounds. Since the alkyne compound is labeled with a stable isotope, various vitamin D compounds labeled with a stable isotope can be produced by the alkyne compound. The vitamin D compound produced using the alkyne compound has excellent storage stability.

2-1. Structure of the alkyne compound of formula (I)

The alkyne compound of formula (I) will be described below.

In formula (I), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of the two ends. That is, the alkyne compound is a compound having an enyne structure. Therefore, it can be said that the present invention provides an enyne compound represented by formula (I).
It should be noted that A is merely a symbol given for the sake of convenience in describing the constituent parts of the vitamin D compound described below, and does not represent the actual elements or the like constituting the alkyne compound of formula (I).

In formula (I), _X1 may be CH or ^13CH , i.e., _X1 forms the alkene portion of the enyne structure.

When X ₁ is CH, at least one of the one or more X ₂ and the one or more X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N.
When _X1 is ^13CH , none of the one or more _X2 and the one or more _X3 may be modified with a stable isotope, or even in this case, at least one of the one or more _X2 and the one or more _X3 may be modified with a stable isotope D, ^13C , ^18O , or ^15N .

In formula (I), _X2 is any one of CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O. m represents the number of _X2 contained in the alkyne compound. m is an integer of 1 to 4. The alkyne compound of formula (I) has m _X2s connected in a linear chain between _X1 and _X3 .
When m is 2 or more, _X2 may be the same or different from each other. That is, when the alkyne compound has 2 or more _X2 , the 2 or more _X2 may be independently selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O.
Preferably, m is 2, 3, or 4, more preferably, m is 3 or 4, and even more preferably, m is 4. The embodiment where m is 4 is further described below with reference to formula (100).

When at least one of the one or more _X2 is modified with a stable isotope D, ^13C , ^18O , or ^15N , neither _X1 nor _X3 may be modified with a stable isotope, or even in this case, either or both of _X1 and _X3 may be modified with a stable isotope.
Provided that none of the one or more _X2s is modified with a stable isotope D, ^13C , ^18O , or ^15N , either or both of _X1 and _X3 may be modified with a stable isotope.

In formula (I), Y is any one _of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent. When two or more Ys are included in formula (I), the Ys may be the same or different from each other. That is, when the alkyne compound has two or more Ys, the two or more Ys may be independently selected from H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _{OSO3Na ,} and a sugar substituent.

In formula (I), R represents an alcohol protecting group, an alkyl group, an alkenyl group, or an aryl group. When two or more R are included in formula (I), the R may be the same or different from each other. That is, when the alkyne compound has two or more R, the two or more R may be independently selected from an alcohol protecting group, an alkyl group, an alkenyl group, and an aryl group.

The alcohol protecting group that the alkyne compound has as R may be, for example, a silyl alcohol protecting group or a benzyl alcohol protecting group. Examples of the silyl alcohol protecting group include, but are not limited to, t-butyldimethylsilyl (also referred to as TBS or TBDMS), triisopropylsilyl (also referred to as TIPS), and triethylsilyl (also referred to as TES). Examples of the benzyl alcohol protecting group include, but are not limited to, p-methoxybenzyl (also referred to as PMB), benzyloxymethyl (also referred to as BOM), and benzyl (also referred to as Bn). The structures of these exemplary alcohol protecting groups are shown in Table 1 below.

The alkyl group that the alkyne compound has as R may be a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkyl group may be, for example, 1 to 8, 1 to 6, or 1 to 4. This carbon number does not include the number of carbon atoms of the substituent.
The alkenyl group that the alkyne compound has as R may be a linear or branched alkyl group having 2 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkenyl group may be, for example, 2 to 8, 2 to 6, or 2 to 4. This carbon number does not include the number of carbon atoms of a substituent.
The aryl group contained as R in the alkyne compound may be a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. The number of carbon atoms in the aryl group is preferably 6 to 18, and may be, for example, 6 to 16, 6 to 14, 6 to 12, or 6 to 10. This number of carbon atoms does not include the number of carbon atoms of the substituent. The aryl group may be an unsubstituted aryl group, and may be, for example, a phenyl group or a naphthyl group.

In formula (I), the sugar substituent may have, for example, the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents, and is an integer of 3 to 5. W may be any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other. That is, the 3 to 5 Ws possessed by the sugar substituent may be independently selected from H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc.

In one embodiment, at least one of the one or more _X2 is any one of CHY, CDY, ¹³ CHY, and ¹³ CDY, for example, any one of the one or more _X2 may be any one of CHY, CDY, ¹³ CHY, and ¹³ CDY.
In this embodiment, preferably, Y may represent any one of H, D, OH, and OR, for example, any one of H, D, and OR. For example, when two or more Y are included in the alkyne compound, the two or more Y may be independently selected from H, D, OH, and OR.

In a particularly preferred embodiment, at least one of the one or more _X2 is CHY or CDY, for example, any of the one or more _X2 may be CHY or CDY.
In this embodiment, preferably, Y may represent any one of H, D, OH, and OR, for example, any one of H, D, and OR. For example, when two or more Y are included in the alkyne compound, the two or more Y may be independently selected from H, D, and OR.
In this embodiment, preferably, the one or more _X2 comprises at least one CDD and at least one CDOR, where the at least one CDOR may be CDO- (an alcohol protecting group), and the alcohol protecting group is as described above.

In formula (I), _X3 is C or ^13C , i.e., _X3 forms the alkyne portion of the enyne structure.

In formula (I), at least one of X ₁ , the one or more X ₂ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N. That is, the alkyne compound of formula (I) is a compound having at least one stable isotope, and the at least one stable isotope may be contained in any one or more of X ₁ , the one or more X ₂ , and X ₃ .

2-2. Structure of the alkyne compound of formula (100)

In a particularly preferred embodiment, in formula (I), m is 4. In this embodiment, the alkyne compound may be a compound represented by the following formula (100). The compound represented by formula (100) is described below.

In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of the two ends.

In formula (100), X ₁ may be CH or ¹³ CH.

When _X1 is CH, at least one of _X21 , _X22 , _X23 , and _X24 and _X3 is modified with a stable isotope D, ^13C , ^18O , or ^15N .
When _X1 is ^13CH , none of _X21 , _X22 , _X23 , and _X24 and _X3 may be modified with a stable isotope, or even in this case, at least one of _X21 , _X22 , _X23 , and _X24 and _X3 may be modified with a stable isotope D, ^13C , ^18O , or ^15N .

In formula (100), X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O. In a preferred embodiment, at least one of X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ is CHY or CDY, and for example, all of X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ may be CHY or CDY.
When at least one of _X21 , _X22 , _X23 , and _X24 is modified with a stable isotope D, ^13C , ^18O , or ^15N , neither _X1 nor X3 may be modified with a stable isotope, or even in this case, either or _both of _X1 and _X3 may be modified with a stable isotope.
When none of _X21 , _X22 , _X23 , and _X24 is modified with a stable isotope D, ^13C , ^18O , or ^15N , either or both of _X1 and _X3 may be modified with a stable isotope.

In formula (100), Y and the R contained in Y may be as described above in formula (I). That is, Y may be any of H, D, _NH2 , ^15NH2 , OH, _18OH , SH, OR, ^O (CO)R, _OSO3H , _OSO3Na , and a sugar substituent. Also, R may represent an alcohol protecting group, an alkyl group, an alkenyl group, or an aryl group.

In formula (100), _X3 is C or ^13C .

In formula (100), at least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N. The alkyne compound of formula (100) is a compound having at least one stable isotope, and the at least one stable isotope may be contained in any one or more of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ .

2-3. How to use alkyne compounds

The alkyne compound according to the present invention may be used to produce a vitamin D compound as described above, and more specifically, may be used as a material for forming the A ring portion of the vitamin D compound.

In one embodiment, a vitamin D compound can be produced by reacting a bromoolefin compound having a CD ring of the vitamin D compound with an alkyne compound according to the present invention. In this reaction, the Br of the bromoolefin compound reacts with the alkyne moiety of the alkyne compound. This produces a vitamin D compound. For a specific example of this reaction, see the Examples below.

The bromoolefin compound may be a compound known in the art. The type of the bromoolefin compound (particularly the side chain portion of the D ring) may be appropriately selected by a person skilled in the art depending on the type of vitamin D compound to be produced.

The hydroxy group of the alkyne compound to be subjected to the reaction may be protected by an alcohol protecting group. In this case, the hydroxy group of the vitamin D compound after the reaction is also protected by an alcohol protecting group. Therefore, a deprotection reaction may be carried out after the reaction. The reagent used for the deprotection reaction may be appropriately selected by a person skilled in the art depending on factors such as the type of alcohol protecting group.

2-4. Method for producing alkyne compounds

The alkyne compound of the present invention can be produced, for example, by using a linear polyhydric alcohol compound as a starting material. The linear polyhydric alcohol compound may be, for example, a polyhydric alcohol compound having at least one hydroxy group or multiple hydroxy groups at each end of the linear chain. In addition, the linear polyhydric alcohol compound may have one or multiple hydroxy groups on one or more of the carbons other than the ends. These hydroxy groups may be protected, for example, by a protecting group known in the art (e.g., the alcohol protecting group described above, etc.).

For example, the method for producing an alkyne compound of the present invention may include a labeling step of labeling a linear polyhydric alcohol compound with deuterium by a deuterium labeling reaction, an alkyne group introduction step of introducing an alkyne structure to one end of the deuterium-labeled polyhydric alcohol, and an alkenyl group introduction step of introducing an alkene structure to the other end of the polyhydric alcohol after the alkyne group introduction step. In this way, the alkyne compound of the present invention can be produced by labeling a linear polyhydric alcohol compound with deuterium, introducing an alkyne group, and introducing an alkenyl group. For specific examples of the production method, see the Examples described below.

The linear polyhydric alcohol compounds are known in the art, and the number of carbon atoms and the position and number of hydroxyl groups in the linear polyhydric alcohol compounds may be appropriately selected depending on the number of carbon atoms in the alkyne compound to be produced and the position and number of hydroxyl groups in the alkyne compound. By adjusting the number of carbon atoms, the position and the number, various alkyne compounds can be produced.

The labeling step may be carried out using a reaction known in the art. The labeling step may include, for example, a deuteration reaction using a predetermined catalyst (e.g., a ruthenium-based catalyst, etc.). FIG. 11 shows natural materials malic acid and aspartic acid as examples of compounds used to obtain the linear polyhydric alcohol compound. As shown in the figure, a deuteration reaction is performed on malic acid or aspartic acid to obtain a deuterated linear polyhydric alcohol. Also, as shown in the figure, a deuteration reaction may be performed on malic acid or aspartic acid labeled with any one or more of ¹³ C, ¹⁵ N, and ¹⁸ O. By such a deuteration reaction, a linear polyhydric alcohol labeled with any one or more of ¹³ C, ¹⁵ N, and ¹⁸ O can be obtained. By carrying out the above-mentioned production method using the linear polyhydric alcohol, an alkyne compound labeled with any one or more of ¹³ C, ¹⁵ N, and ¹⁸ O can be obtained.
Furthermore, a deuteration reaction may be carried out on malic acid or aspartic acid labeled with multiple ¹³ C, multiple ¹⁵ N ^, or multiple ¹⁸ O. By such a deuteration reaction, a linear polyhydric alcohol labeled with multiple 13 C, multiple ¹⁵ N, or multiple ¹⁸ O can be obtained. By carrying out the above-mentioned production method using the linear polyhydric alcohol, an alkyne compound labeled with multiple ¹³ C, multiple ¹⁵ N, or multiple ¹⁸ O can be obtained.
As described above, various types of stable isotope-labeled starting materials can be obtained, and an alkyne compound corresponding to each starting material can be obtained.

The alkyne group introduction step may be carried out using a reaction known in the art. For example, the alkyne group introduction step may include a terminal alkyne introduction reaction in which an alkyne group is introduced at one end using trimethylsilylacetylene.

The alkenyl group introduction step may be carried out using a reaction known in the art. For example, the alkenyl group introduction step may include a cyano group introduction reaction to introduce a cyano group to the other end, and a reaction to convert the cyano group into an alkenyl group.

3. Second embodiment (vitamin D compound)

The present invention provides a vitamin D compound represented by the following formula (III). The vitamin D compound is labeled with a stable isotope as described below. The vitamin D compound may be used, for example, in mass spectrometry, for example, for mass spectrometry in which quantification is performed by isotope dilution. The mass spectrometry may be for detecting and/or quantifying vitamin D metabolites. The vitamin D compound according to the present invention also has the advantage of having excellent storage stability.

3-1. Structure of vitamin D compound of formula (III)

The vitamin D compounds of formula (III) are described below.

In formula (III), A' represents a cyclic carbon chain, which may represent the cyclic carbon chain corresponding to the A ring of a vitamin D compound.
It should be noted that A', seco-B, C, and D are merely symbols given for the sake of convenience to explain the constituent parts of the vitamin D compound, and do not represent the actual elements or the like constituting the vitamin D compound of formula (III).

In formula (III), _X'1 may be C or ¹³ C. When the vitamin D compound of formula (III) is prepared using the alkyne compound of formula (I), the carbon of _X1 in formula (I) becomes the carbon of _X'1 in formula (III).

When _X'1 is C, at least one of the one or more _X'2 and _X'3 is modified with a stable isotope D, ^13C , ^18O , or ^15N .
When X' ₁ is ¹³ C, none of the one or more X' ₂ and the one or more X' ₃ may be modified with a stable isotope, or even in this case, at least one of the one or more X' ₂ and the one or more X' ₃ may be modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N.

In formula (III), X' ₂ is any one of CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O. The vitamin D compound of formula (III) has m X' ₂ connected in a linear chain between X' ₁ and X' _3. m is any integer from 1 to 4. When m is 2 or more, X' ₂ may be the same or different from each other. That is, when the vitamin D compound has two or more X' ₂ , the two or more X' ₂ may be independently selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O.
Preferably, m is 2, 3, or 4, more preferably, m is 3 or 4, and even more preferably, m is 4. The embodiment where m is 4 is further described below with reference to formula (300).
When the vitamin D compound of formula (III) is prepared using the alkyne compound of formula (I), the carbon _X2 of formula (I) becomes the carbon _X'2 of formula (III).

When at least one of the one or more _X'2 is modified with a stable isotope D, ^13C , ^18O , or ^15N , neither of the _X'1 nor the _X'3 may be modified with a stable isotope, or even in this case, either or both of the _X'1 and the _X'3 may be modified with a stable isotope.
Provided that none of the one or more _X'2 is modified with a stable isotope D, ^13C , ^18O , or ^15N , either or both of the _X'1 and the _X'3 may be modified with a stable isotope.

In formula (III), Y' is any _one of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent. When two or more Y' are included in formula (III), Y' may be the same or different from each other. That is, when the alkyne compound has two or more Y', the two or more Y' may be independently selected ^{from H, D, NH2, 15NH2, OH, 18OH} _{, SH} ^, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent.
When the vitamin D compound of formula (III) is produced using the alkyne compound of formula (I), Y in formula (I) corresponds to Y' in formula (III).

In formula (III), R' represents an alkyl group, an alkenyl group, or an aryl group. When two or more R' are included in formula (III), the R' may be the same or different from each other. That is, when the alkyne compound has two or more R', the two or more R' may be independently selected from among alkyl groups, alkenyl groups, and aryl groups.

When a vitamin D compound of formula (III) is produced using an alkyne compound of formula (I) containing OR as Y and having an alcohol protecting group as R of the OR, the alcohol protecting group of the OR of the alkyne compound of formula (I) may be deprotected and exist as OH in the vitamin D compound of formula (III).

The alkyl group that the vitamin D compound has as R' may be a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkyl group may be, for example, 1 to 8, 1 to 6, or 1 to 4. This carbon number does not include the carbon atoms of any substituents.
The alkenyl group that the alkyne compound has as R' may be a linear or branched alkyl group having 2 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkenyl group may be, for example, 2 to 8, 2 to 6, or 2 to 4. This carbon number does not include the number of carbon atoms of a substituent.
The aryl group contained as R' in the alkyne compound may be a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. The number of carbon atoms in the aryl group is preferably 6 to 18, and may be, for example, 6 to 16, 6 to 14, 6 to 12, or 6 to 10. This number of carbon atoms does not include the number of carbon atoms of the substituent. The aryl group may be an unsubstituted aryl group, and may be, for example, a phenyl group or a naphthyl group.

In formula (III), the sugar substituent may have, for example, the structure:
Wo in the sugar substituent represented by this structure is the same as Wo described above in relation to formula (I), and the same description also applies to Wo that may be included in formula (III).

In one embodiment, at least one of the one or more _X'2 is any one of CHY', CDY', ¹³ CHY', and ¹³ CDY', for example, any one of the one or more _X'2 may be any one of CHY', CDY', ¹³ CHY', and ¹³ CDY'.
In this embodiment, preferably, Y' may represent any of H, D, OH, and OR', for example, any of H, D, OH, and OR'. For example, when two or more Y' are included in the vitamin D compound, the two or more Y' may be independently selected from H, D, OH, and OR'.

In a particularly preferred embodiment, at least one of the one or more _X'2 is CHY' or CDY', for example any of the one or more _X'2 may be CHY' or CDY'.
In this embodiment, preferably, Y' may represent any one of H, D, and OH, for example, any one of H, D, and OH. For example, when two or more Y' are included in the alkyne compound, the two or more Y' may be independently selected from H, D, and OH.
In this embodiment, preferably, the one or more _X'2 comprises at least one CDD and at least one CDOH.

In formula (III), _X'3 is C or ¹³ C. When the vitamin D compound of formula (III) is produced using the alkyne compound of formula (I), the carbon of _X3 in formula (I) corresponds to the carbon of _X'3 in formula (III).

As shown in formula (III), X' ₁ and X' ₃ may be bonded together, i.e., a ring structure may be formed by the X' ₁ , the one or more X' ₂ , and the X' _3. The ring structure corresponds to the A ring in the vitamin D compound.

In formula (III), at least one of X' ₁ , the one or more X' ₂ , and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N. That is, the vitamin D compound of formula (III) is a compound having at least one stable isotope, and the at least one stable isotope may be contained in any one or more of X' ₁ , the one or more X' ₂ , and X' ₃ .

The vitamin D compound has a seco-B ring, a C ring, and a D ring, as shown in formula (III). The D ring may have P bonded thereto, which represents a side chain.

In formula (III), P may be a _C1 - _C10 alkyl group, which may or may not be substituted with a hydroxy group, or a C2- _C10 alkenyl group, which may or may not be substituted with a _hydroxy group. The alkyl or alkenyl group may have a lactone group substituted with a hydroxy group.

In one embodiment, P in formula (III) is
It may be expressed as:
where Q, Q', Q'', Q''', and Q'''' each independently represent any of H, CH ₂ , CH ₃ , C ₂ H ₅ , O, OH, and a sugar substituent.

More specific examples of P include the following: P may be any one of these.

The structure of P need not be limited to those listed above, and any of the D-ring side chains of vitamin D compounds known in the art may be employed as the structure of P.

3-2. Structure of vitamin D compound of formula (200)

In a particularly preferred embodiment, in formula (III), m is 4. In this embodiment, the alkyne compound may be a compound represented by the following formula (200). The compound represented by formula (200) is described below.

In formula (200), A' represents a cyclic carbon chain. A' is the portion that corresponds to the A ring of a vitamin D compound.

In formula (200), X' ₁ may be C or ¹³ C. When the vitamin D compound of formula (200) is prepared using an alkyne compound of formula (100), the carbon of X ₁ in formula (100) becomes the carbon of X' ₁ in formula (200).

When _X'1 is C, at least one of the one or more _X'2 and _X'3 is modified with a stable isotope D, ^13C , ^18O , or ^15N .
When X' ₁ is ¹³ C, none of the one or more X' ₂ and the one or more X' ₃ may be modified with a stable isotope, or even in this case, at least one of the one or more X' ₂ and the one or more X' ₃ may be modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N.

In formula (200), X' ₂₁ , X' ₂₂ , X' ₂₃ , and X' ₂₄ each independently represent any one selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O, and ¹³ C ¹⁸ O. In a preferred embodiment, at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ , and X' ₂₄ is CHY' or CDY', and for example, all of X' ₂₁ , X' ₂₂ , X' ₂₃ , and X' ₂₄ may be CHY or CDY.
When the vitamin D compound of formula (200) is prepared using the alkyne compound of formula (100), the carbons X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ of formula (100) correspond to the carbons X' ₂₁ , X' ₂₂ , X' ₂₃ , and X' ₂₄ of formula (200).

When at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N, neither X' ₁ nor X' ₃ may be modified with a stable isotope, or even in this case, either or both of X' ₁ and X' ₃ may be modified with a stable isotope.
When none of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N, either or both of X' ₁ and X' ₃ may be modified with a stable isotope.

In formula (200), Y' and R' contained in Y' may be as described above in formula (III). That is, Y' may be any one of H, D, NH2, _15NH2 , OH, ^18OH , SH _, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent. R' represents ^an alkyl group, an alkenyl group, or an aryl group.

In one embodiment, at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is any one of CHY', CDY', ¹³ CHY' and ¹³ CDY', for example, all of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ may be any one of CHY', CDY', ¹³ CHY' and ¹³ CDY'.
In this embodiment, preferably, Y' may represent any of H, D, OH, and OR', for example, any of H, D, OH, and OR'. For example, when two or more Y' are included in the vitamin D compound, the two or more Y' may be independently selected from H, D, OH, and OR'.

In a particularly preferred embodiment, at least one of the one or more _X'2 is CHY' or CDY', for example any of the one or more _X'2 may be CHY' or CDY'.
In this embodiment, preferably, Y' may represent any one of H, D, and OH, for example, any one of H, D, and OH. For example, when two or more Y' are included in the alkyne compound, the two or more Y' may be independently selected from H, D, and OH.
In this embodiment, preferably, the one or more _X'2 comprises at least one CDD and at least one CDOH.

In formula (200), _X'3 is C or ¹³ C. When the vitamin D compound of formula (200) is produced using the alkyne compound of formula (100), the carbon of _X3 in formula (100) corresponds to the carbon of _X'3 in formula (200).

As shown in formula (200), X' ₁ and X' ₃ may be bonded together, i.e., a ring structure may be formed by X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , and X' ₂₄ , and X' _3. The ring structure corresponds to the A ring in vitamin D compounds.

In formula (200), at least one of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ , and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N. That is, the vitamin D compound of formula (200) is a compound having at least one stable isotope, and the at least one stable isotope may be contained in any one or more of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ , and X' ₃ .

P in formula (200) is as explained above in formula (III), and the explanation also applies to formula (200).

The vitamin D compounds of the present invention include 25-hydroxyvitamin D ₃ (25OHD ₃ ), 3-epi-25-hydroxyvitamin D ₃ (3-epi-25OHD ₃ ), 25-hydroxyvitamin D ₂ (25OHD ₂ ), 24R,25-dihydroxyvitamin D ₃ (24R,25(OH) ₂ D ₃ ), 23S,25-dihydroxyvitamin D ₃ (23S,25(OH) ₂ D ₃ ), 1α,25-dihydroxyvitamin D ₃ (1α,25(OH) ₂ D ₃ ), 25-hydroxyvitamin D _3-26R ,23S-lactone (25OHD ₃ -26R,23S-lactone), 1α,25-dihydroxyvitamin D _3-26R ,23S-lactone (1α,25(OH) ₂ D _3-26R ,23S-lactone), 1α,25-dihydroxyvitamin D ₂ (1α,25(OH) ₂ D ₂ ), 1α,24R,25-trihydroxyvitamin D ₃ (1α,24R,25(OH) ₃ D ₃ ), or 4β,25-dihydroxyvitamin D ₃ (4β,25(OH) ₂ D ₃ ). The chemical structures of these compounds are shown in FIG. 10. That is, the vitamin D compound of the present invention may be a vitamin D compound in which the A-ring portion of the chemical structure of each compound shown in the figure is labeled with a stable isotope according to the present invention. In one embodiment, the vitamin D compound of the present invention may be a vitamin D compound in which at least one of the hydrogen atoms bonded to the carbon atoms forming the A-ring portion of the chemical structure of each compound shown in the figure is deuterium. The number of deuterium atoms bonded to the carbon atoms forming the A-ring portion may be, for example, 2 or more, particularly 3 or more. The number of deuterium atoms bonded to the carbon atoms forming the A ring moiety may be, for example, 7 or less, particularly 6 or less, and more particularly 5 or less.

3-3. How to use vitamin D compounds

The vitamin D compounds of the present invention may be used in various analytical methods, in particular in mass spectrometry, for example in isotope dilution mass spectrometry. The mass spectrometry method may more particularly be LC-MS/MS or LC-MS. By carrying out the analytical method using the vitamin D compounds of the present invention, it is possible to detect or quantify vitamin D compounds (e.g. vitamin D metabolites in a biological sample) that have the same chemical structure but are not isotopically labeled.

The vitamin D compound of the present invention may be used, for example, as a standard substance in the above-mentioned analytical method, and may in particular be used as an internal standard substance.

In the mass spectrometry method, the vitamin D compound of the present invention may be derivatized. The derivatization can improve the detection accuracy. For the derivatization, a derivatization reagent known in the art may be used.

In the mass spectrometry method, the vitamin D compound of the present invention or the derivatized vitamin D compound may be ionized. The ionization may be, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI) for more efficient ionization, but may also be ionized by other ionization methods. The ionization allows for more accurate detection or quantification.

The mass spectrometry method may be performed, for example, by a liquid chromatography tandem mass spectrometer (LC-MS/MS) or a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS), but the apparatus for performing the mass spectrometry step is not limited to these. The specific steps in the mass spectrometry step can be appropriately set by a person skilled in the art depending on, for example, the ionization method employed and the apparatus used.

3-4. Manufacturing method of vitamin D compounds

The vitamin D compound of the present invention may be produced by reacting the alkyne compound of the present invention with a compound for forming a CD ring of the target vitamin D compound. The compound for forming a CD ring may be an olefin compound having a CD ring. As the olefin compound, a compound known in the art may be used. The olefin compound may be, for example, a compound having a structure represented by the following formula (II). As the compound, a compound known in the art may be used.
In the compound of formula (II), C and D represent the moieties which form the C-ring and D-ring, respectively, when the vitamin D compound is synthesized.
P in the compound of formula (II) may be, for example, the same as P described above in formula (III), but the hydroxy group contained in P may be protected by the alcohol protecting group described above in the compound of formula (II). The alcohol protecting group may be, for example, TES.
Z in the compound of formula (II) may be a halogen or pseudohalogen group, for example representing F, Cl, Br, I, or OTf. In one embodiment, Z is Br, i.e. the compound of formula (II) may be a bromoolefin compound.

That is, the method for producing the vitamin D compound of the present invention may include reacting an alkyne compound of formula (I) with a compound of formula (II). More specifically, the reaction may include a reaction between an alkynyl group in the alkyne compound of formula (I) and Z in the compound of formula (II). By the reaction, a bond is formed between the carbon bonded to Z in the compound of formula (II) and the carbon other than _X3 , out of the two carbons constituting the alkynyl group in the alkyne compound of formula (I), to form a seco-B ring. Furthermore, a bond is formed between _X1 and _X3 in the alkyne compound of formula (I) to form an A' ring. In this way, the A' ring and the seco-B ring are formed, and the vitamin D compound of the present invention is formed.

4. Third embodiment (analysis method using vitamin D compounds)

4-1. Examples of analysis methods

The present invention also provides an analytical method using a vitamin D compound according to the present invention. The analytical method may include analyzing a sample by mass spectrometry, as described in 3-3 above. The sample may be a sample that contains, or may contain (i.e., may not contain), a vitamin D compound having the same chemical structure as the vitamin D compound according to the present invention, but not isotopically labeled, as a target for detection or quantification, for example. The sample may be, for example, a biological sample, and in particular a body fluid or a sample derived from a body fluid, and more specifically, may be any of serum, plasma, blood, urine, cerebrospinal fluid, and saliva.

According to one embodiment, the vitamin D compound of the present invention may be used as an internal standard in the analytical method. The method of using the vitamin D compound of the present invention as an internal standard may be a method known in the art. For example, in the analytical method, the amount or concentration of the compound to be analyzed (e.g., a vitamin D metabolite) may be determined based on the relationship between the peak area ratio and the concentration ratio of the vitamin D compound of the present invention.

4-2. Modifications to the analysis method

In a modified example of the analysis method described in 4-1 above, vitamin D compounds that do not have a hydroxy group at position 1 of the A ring may be analyzed separately from vitamin D compounds that have a hydroxy group at position 1 of the A ring. More specifically, the vitamin D compounds that do not have a hydroxy group at position 1 of the A ring may include at least one of 2-hydroxyvitamin D compounds and 4-hydroxyvitamin D compounds. Even more specifically, the vitamin D compounds that do not have a hydroxy group at position 1 of the A ring may include at least one of 4β,25-dihydroxyvitamin D3 compounds and 4-hydroxyvitamin D compounds. This modified example will be described in more detail below.

As mentioned above, there is a high demand for detection or quantification of vitamin D metabolites in biological samples in clinical and food nutritional aspects. Among various vitamin D metabolites, one of the most important metabolites in terms of physiological activity is 1α,25-dihydroxyvitamin _D3 , also known as active vitamin D, and its selective extraction is extremely important. LC-MS/MS has high separation ability by combining chromatographic separation and separation by m/z (mass-to-charge ratio) of fragment ions, and the sensitivity can be further increased by using derivatization reagents.

However, there are cases where two or more vitamin D metabolites with different chemical structures cannot be individually detected/quantified by LC-MS/MS alone. For example, there are cases where two or more positional isomers that differ only in the position of the hydroxyl group cannot be individually detected/quantified by LC-MS/MS alone. An example of such a case will be described with reference to FIG. 6.
As shown in the figure, it is assumed that the sample to be analyzed contains three _types of vitamin D compounds: 1α,25-dihydroxyvitamin _D3 (1α,25(OH) _2D3 ), 24R,25-dihydroxyvitamin _D3 (24R,25(OH) _2D3 ) _, and 4β,25-dihydroxyvitamin _D3 (4β,25(OH _{) 2D3} ).
When the sample is derivatized using, for example, the derivatization reagent DAP-PA shown in the figure, three derivatized compounds are generated as shown on the right side of the figure. These three derivatives are cleaved at the positions indicated by the arrows in the figure. As can be seen from the products after the cleavage, 24R,25-dihydroxyvitamin _D3 can be separated from 1α,25-dihydroxyvitamin _D3 by the m/z of the fragment ions, whereas 4β,25-dihydroxyvitamin _D3 cannot be separated in this manner. Furthermore, 1α,25-dihydroxyvitamin _D3 and 4β,25-dihydroxyvitamin _D3 are difficult to separate by chromatography.

By carrying out a predetermined separation process and then carrying out an analytical process using a vitamin D compound according to the present invention on the sample obtained by the separation process, it is possible to individually detect or quantify two or more vitamin D compounds that are difficult to separate as described above. The present invention also provides an analytical method for selectively detecting or quantifying two or more vitamin D compounds that are difficult to separate by LC-MS/MS alone. In other words, the analytical method of the present invention also provides an analytical method for individually detecting or quantifying two or more positional isomers of a vitamin D compound. As described above, the two or more positional isomers may be positional isomers in which the position of the hydroxyl group bonded to the A ring is different from each other. The two or more positional isomers may have the same molecular weight.

In one embodiment, the analytical method of the present invention comprises:
A fractionation step of fractionating the sample into at least one first fraction in which vitamin D compounds having a hydroxy group at position 1 of the A ring are recovered and at least one second fraction in which vitamin D compounds having a hydroxy group at position 1 of the A ring are not recovered;
Analysing either or both of said at least one first fraction and said at least one second fraction using a vitamin D compound of the present invention.
In this embodiment, the second fraction may include vitamin D compounds that do not have a hydroxy group at the 1-position of the A ring.
The sample does not necessarily need to contain a vitamin D compound having a hydroxy group at position 1 of the A ring. That is, the first fraction may be a fraction obtained by performing an operation for recovering a vitamin D compound having a hydroxy group at position 1 of the A ring, and the fraction does not necessarily need to contain the vitamin D compound.
The fractionation step and the analysis step will be described below.

4-2-1. Fractionation Step The sample to be subjected to the fractionation step may be the sample described in 4-1 above.

The vitamin D compound having a hydroxy group at position 1 of the A ring may be, for example, a vitamin D compound having the structure shown on the left of FIG. 7, i.e., a 1-hydroxyvitamin D compound. As shown in the figure, the 1-hydroxyvitamin D compound has a hydroxy group at position 1 of the A ring. Furthermore, the 1-hydroxyvitamin D compound may have a hydroxy group at position 3 of the A ring. The P contained in the 1-hydroxyvitamin D compound may be as described above with respect to formula (III).

The vitamin D compound not having a hydroxy group at position 1 of the A ring may be a vitamin D compound having the structure shown on the right of FIG. 7, i.e., a 2-hydroxyvitamin D compound or a 4-hydroxyvitamin D compound. The 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound has a hydroxy group at position 2 or 4 of the A ring as shown in the figure. Furthermore, the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may have a hydroxy group at position 3 of the A ring. The P contained in the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may be as described above with respect to formula (III).

In particular, the 1-hydroxyvitamin D compound and the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may be the same except for the difference in the position of the hydroxy group attached to the A ring.
The 1-hydroxyvitamin D compound may be, for example, 1α,25-dihydroxyvitamin _D3 , as shown on the left side of Figure 8. The 4-hydroxyvitamin D compound may be, for example, 4β,25-dihydroxyvitamin _D3 , as shown on the right side of the figure.
That is, the sample to be subjected to the above-mentioned analytical method may be a sample that can contain 1α,25-dihydroxyvitamin _D3 and/or 4β,25-dihydroxyvitamin _D3 as the subject of analysis.

The sample subjected to the above-mentioned analytical method does not necessarily need to contain the vitamin D compound to be analyzed; that is, the analysis may reveal that the sample does not contain the vitamin D compound to be analyzed.

The fractionation step may be carried out, for example, using a capture substance that selectively captures (e.g., binds or adsorbs) only vitamin D compounds having a hydroxy group at position 1 of the A ring. Examples of the capture substance include, but are not limited to, antibodies or aptamers. Reagents known in the art may be used as the capture substance, and a person skilled in the art can select the capture substance appropriately depending on the vitamin D compound to be captured.

In one embodiment, in the fractionation step, the sample may be applied to a column having an antibody that selectively captures only vitamin D compounds having a hydroxy group at position 1 of the A ring. An example of the column is an antibody column, such as 1,25-(OH) _2- Vitamin _D3 / _D2 -ImmuTube manufactured by Immundiagnostik AG.
The sample is added to the column, and the antibody in the column captures vitamin D compounds having a hydroxy group at position 1 of the A ring. The uncaptured vitamin D compounds not having a hydroxy group at position 1 of the A ring are then recovered from the column to obtain the second fraction. The captured vitamin D compounds having a hydroxy group at position 1 of the A ring are then recovered from the column by a predetermined elution process to obtain the first fraction.

4-2-2. Analysis process

In the analysis step, either or both of the at least one first fraction and the at least one second fraction are analyzed using the vitamin D compound of the present invention. The analysis may be performed by the mass spectrometry method described in 4-1 above.

For example, the second fraction may be subjected to mass spectrometry using, as a standard substance, a vitamin D compound of the present invention corresponding to a target vitamin D compound expected to be contained in the second fraction (a compound having a chemical structure in which the A ring of the target vitamin D compound is labeled with a stable isotope). As an example, when the target vitamin D compound is 4β,25-dihydroxyvitamin _D3 , LC-MS/MS may be performed using, as an internal standard substance, a compound having a chemical structure in which the A ring of _4β ,25-dihydroxyvitamin D3 is labeled with a stable isotope.

Furthermore, mass spectrometry may be performed on the first fraction using, as a standard substance, a vitamin D compound of the present invention corresponding to a target vitamin D compound expected to be contained in the first fraction (a compound having a chemical structure in which the A ring of the target vitamin D compound is labeled with a stable isotope). For example, when the target vitamin D compound is 1α,25-dihydroxyvitamin _D3 , LC-MS/MS may be performed using, as an internal standard substance, a compound having a chemical structure in which the A ring of 1α,25-dihydroxyvitamin _D3 is labeled with a stable isotope.

The above analytical method makes it possible to selectively detect or quantify two or more vitamin D compounds that are difficult to separate using LC-MS/MS alone.

For example, the analytical method can selectively recover 2(or 4)-hydroxyvitamin D compounds, and the recovered 2(or 4)-hydroxyvitamin D compounds can be analyzed by LC-MS/MS (liquid chromatography-tandem mass spectrometry). The analytical method can be used regardless of the mass spectrometer manufacturer.
Among them, 4β,25-dihydroxyvitamin D ₃ is a positional isomer of 1α,25-dihydroxyvitamin D ₃ , which is called active vitamin D and is physiologically the most important, and has the same molecular weight and cleavage pattern in MS/MS and almost the same HPLC chromatographic behavior, making it extremely difficult to separate. For this reason, 4β,25-dihydroxyvitamin D ₃ has been hidden behind 1α,25-dihydroxyvitamin D ₃ , and its physiological role has been unknown. It is also possible that the phenomenon that was previously considered to be the activity of 1α,25-dihydroxyvitamin D ₃ may be due to 4β,25-dihydroxyvitamin D _3. The analytical method of the present invention can separate and quantify 4β,25-dihydroxyvitamin D _{3 ,} which is useful for evaluating the function or activity of 4β,25-dihydroxyvitamin D ₃ and is also useful for more accurately evaluating the function or activity of 1α,25-dihydroxyvitamin D 3. In this way, it is considered that the analytical method of the present invention will greatly contribute to elucidating the mechanism of action of vitamin D compounds.

5. Fourth embodiment (method for producing vitamin D compound)

The present invention also provides a method for producing the vitamin D compound of the present invention. The production method may be carried out as described in 3-4 above. That is, in the production method of the present invention, the alkyne compound represented by the formula (I) may be used as a material for forming the A ring of the vitamin D compound. An example of a chemical reaction formula in this case is shown in FIG. 9A. As shown in the figure, in the production method of the present invention, the vitamin D compound represented by the formula (III) may be produced by reacting the alkyne compound represented by the formula (I) with a compound having a structure represented by the formula (II).

In a preferred embodiment, in the manufacturing method of the present invention, the alkyne compound represented by the formula (100) may be used as a material for forming the A ring of the vitamin D compound. In this case, an example of a chemical reaction formula is shown in FIG. 9B. As shown in the figure, in the manufacturing method of the present invention, the vitamin D compound represented by the formula (200) may be produced by reacting the alkyne compound represented by the formula (100) with a compound having a structure represented by the formula (II).

6. Examples The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

6-1. Example 1: Production of alkyne compounds and vitamin D compounds

(1) Synthesis of alkyne compound 10

Alkyne compound 10 was prepared by the synthetic route shown below, with each reaction in the synthetic route being explained below.

(1-1) Synthesis of d ₃ -diol 2
Under a hydrogen atmosphere, diol 1 (4.0 g, 21.0 mmol) was dissolved in deuterium oxide (80 mL), ruthenium carbon (4.0 g) was added at room temperature, and the mixture was stirred at 80° C. for 24 hours. After stirring, the reaction solution was filtered through Celite (ethyl acetate/methanol = 8/1) and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain d ₃ -diol 2 (3.84 g, 96% yield) as a colorless liquid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 4.34-4.42 (m, 1H), 4.12-4.17 (m, 1H), 1.68-1.81 (m, 2H), 1.19 (s, 9H).
HRMS (ESI, M+Na) calcd. for C ₉ H ₁₅ D ₃ O ₄ Na 216.1291, found 216.1268.

(1-2) Synthesis of d ₃ -tosylate 3
Under an argon atmosphere, d ₃ -diol 2 (7.8 g, 40.4 mmol) was dissolved in pyridine (80.8 mL), tosyl chloride (8.08 mL, 42.4 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After stirring, distilled water (80 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3/1) to obtain d ₃ -tosylate 3 (7.05 g, 50% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.80 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.23-4.29 (m, 1H), 4.09-4.16 (m, 1H), 2.45 (s, 3H), 1.76-1.83 (m, 1H), 1.67-1.73 (m, 1H), 1.18 (s, 9H).
HRMS (ESI, M+Na) calcd. for C ₁₆ H ₂₁ D ₃ O ₆ SNa 370.1380, found 370.1413.

(1-3) Synthesis of d ₃ -epoxide 4
Under an argon atmosphere, _d3 -tosylate 3 (10.8 g, 31.1 mmol) was dissolved in dehydrated dimethylformamide (156 mL), and sodium hydride (1.49 g, 37.3 mmol) was added under ice cooling and stirred at room temperature for 0.5 hours. After stirring, distilled water (100 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 8/1) to obtain _d3 -epoxide 4 (3.96 g, 73% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.22 (t, J = 6.0 Hz, 2H), 1.80-1.94 (m, 2H), 1.20 (s, 9H).
HRMS (ESI, M+Na) calcd. for C ₉ H ₁₃ D ₃ O ₃ Na 198.1185, found 198.1213.

(1-4) Synthesis of _d3 -alkyne 5

Under an argon atmosphere, trimethylsilylacetylene (6.4 mL, 45.6 mmol) was dissolved in anhydrous tetrahydrofuran solution (24.8 mL), and n-butyllithium (1.6 M hexane solution) (13.7 mL, 35.6 mmol) was added dropwise at -78°C to prepare a reaction mixture. After stirring the reaction mixture at the same temperature for 1 hour, a tetrahydrofuran solution of d ₃ -epoxide 4 (3.47 g, 19.8 mmol) and boron trifluoride diethyl ether complex (3.23 mL, 25.7 mmol) were added dropwise in that order. The reaction mixture was stirred at the same temperature for another 30 minutes. A saturated aqueous ammonium chloride solution (20 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=10/1) to obtain d ₃ -alkyne 5 (3.33 g, yield 61%) as a yellow liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.27-4.33 (m, 1H), 4.15-4.21 (m, 1H), 1.88-1.95 (m, 1H), 1.75-1.81 (m, 1H), 1.18 (s, 9H), 0.15 (s, 9H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₂₃ D ₃ O ₃ SiNa 296.1737, found 296.1732.

(1-5) Synthesis of _d3 -alkyne 6
Under an argon atmosphere, _d3 -alkyne 5 (3.2 g, 11.7 mmol) was dissolved in dehydrated dimethylformamide (23.4 mL), and imidazole (2.15 g, 31.6 mmol), tert-butyldimethylsilyl chloride (3.50 g, 23.4 mmol), and 4-dimethylaminopyridine (143 mg, 1.17 mmol) were added and stirred at room temperature for 3 hours. After stirring, distilled water (23 mL) was added to the reaction solution, which was extracted with a hexane/ethyl acetate mixed solvent. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 10/1) to obtain _d3 -alkyne 6 (4.61 g, 100% yield) as a yellow liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.19-4.25 (m, 1H), 4.05-4.13 (m, 1H), 1.93-2.00 (m, 1H), 1.73-1.79 (m, 1H), 1.22 (s, 9H), 0.88 (s, 9H), 0.14 (s, 9H), 0.08 (d, J = 10.5 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₂₀ H ₃₇ D ₃ O ₃ Si ₂ Na 410.2602, found 410.2578.

(1-6) Synthesis of _d3 -alkyne 7
Under an argon atmosphere, _d3 -alkyne 6 (4.51 g, 11.6 mmol) was dissolved in methanol (107 mL), and sodium methoxide (5.0 M methanol solution) (9.28 mL, 46.4 mmol) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 24 hours. After stirring, a saturated aqueous solution of ammonium chloride (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 30/1) to obtain _d3 -alkyne 7 (2.32 g, 86% yield) as a yellow liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 3.79-3.85 (m, 1H), 3.71-3.77 (m, 1H), 1.99 (s, 1H), 1.91-1.97 (m, 1H), 1.77-1.84 (m, 1H), 0.89 (s, 9H), 0.11 (s, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₂ H ₂₁ D ₃ O ₂ SiNa 254.1632, found 254.1631.

(1-7) Synthesis of _d3 -alkyne 8
Under an argon atmosphere, _d3 -alkyne 7 (1.0 g, 4.32 mmol) was dissolved in methylene chloride (14 mL), triethylamine (2.1 mL, 15.1 mmol), tosyl chloride (1.65 g, 8.64 mmol), and 4-dimethylaminopyridine (52 mg, 0.43 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Distilled water (20 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 20:1) to give _d3 -alkyne 8 (1.67 g, 99% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.79 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 4.13 (dd, J = 7.3, 5.5 Hz, 2H), 2.45 (s, 3H), 2.06-1.94 (m, 2H), 1.82 (dt, J = 14.2, 5.5 Hz, 1H), 0.82 (s, 9H), 0.02 (d, J = 22.9 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₉ H ₂₇ D ₃ O ₄ SSiNa 408.1720, found 408.1677.

(1-8) Synthesis of _d3 -alkyne 9
Under an argon atmosphere, _d3 -alkyne 8 (1.67 g, 4.27 mmol) was dissolved in dimethyl sulfoxide (14 mL), sodium cyanide (314 mg, 6.41 mmol) was added at room temperature, and the mixture was stirred at 90°C for 30 minutes. After stirring, distilled water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 20:1) to obtain _d3 -alkyne 9 (1.0 g, 100% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 2.47-2.37 (m, 2H), 2.08-1.95 (2H), 1.94-1.80 (m, 1H), 0.89 (s, 9H), 0.11 (d, J = 1.4 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₃ H ₂₀ D ₃ NOSiNa 263.1635, found 263.1632.

(1-9) Synthesis of _d3 -alkyne 10
Under an argon atmosphere, _d3 -alkyne 9 (506 mg, 2.09 mmol) was dissolved in dehydrated methylene chloride (11 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (2.5 mL, 2.50 mmol) was added dropwise under ice cooling and stirred for 1 hour. After stirring, isopropanol (1.75 mL), silica gel, and distilled water were added to the reaction solution, and the mixture was stirred for 30 minutes, filtered, and the solvent was removed by distillation. After removing the solvent, the residue (containing _d3 -aldehyde) was used in the next step without further purification.
Under an argon atmosphere, methyltriphenylphosphonium iodide (2.79 g, 6.90 mmol) was dissolved in anhydrous tetrahydrofuran solution (15 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (3.2 mL, 6.15 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then a tetrahydrofuran solution of d ₃ -aldehyde (599 mg, 2.46 mmol) was added dropwise. The reaction mixture was stirred at the same temperature for another hour. After the stirring, distilled water (20 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. The residue was purified by silica gel column chromatography (hexane) to obtain d ₃ -alkyne 10 (461 mg, 61% yield, 2 steps) as a colorless liquid. The d ₃ -alkyne 10 is also referred to as "alkyne compound 10" in this specification. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.89-5.76 (m, 1H), 5.03 (dd, J = 16.9, 1.8 Hz, 1H), 4.96 (d, J = 10.1 Hz, 1H), 2.21-2.00 (m, 2H), 1.97 (s, 1H), 1.79-1.66 (m, 1H), 1.66-1.58 (m, 1H), 0.89 (s, 9H), 0.07 (d, J = 4.6 Hz, 6H).

(2) Synthesis of alkyne compound 17

Alkyne compound 17 was produced by the synthetic route shown below. As shown in the synthetic route, alkyne compound 17 was synthesized using _d3 -alkyne 7 described in (1) above. Each reaction in the synthetic route is described below.

(2-1) Synthesis of _d3 -alkyne 11

Under an argon atmosphere, _d3 -alkyne 7 (0.80 g, 3.46 mmol) was dissolved in dimethyl sulfoxide (34.6 mL), 2-iodoxybenzoic acid (1.93 g, 6.91 mmol) was added, and the mixture was stirred at 50°C for 1.5 hours. After stirring, a saturated aqueous solution of sodium thiosulfate (34.6 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 50/1) to obtain _d3 -alkyne 11 (0.57 g, 72% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 9.82 (t, J = 1.8 Hz, 1H), 2.77-2.64 (m, 2H), 2.02 (s, 1H), 0.86 (d, J = 2.7 Hz, 9H), 0.09 (d, J = 13.3 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₂ H ₁₉ D ₃ O ₂ SiNa 252.1475, found 252.1503.

(2-2) Synthesis of _d3 -alkyne 12

Under an argon atmosphere, _d3 -alkyne 11 (0.55 g, 2.41 mmol) was dissolved in dehydrated methylene chloride (24.1 mL), and (ethoxycarbonylmethylene)triphenylphosphorane (1.68 g, 4.83 mmol) was added under ice cooling and stirred for 1 hour. After stirring, distilled water (24.1 mL) was added to the reaction solution, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 100/1) to obtain _d3 -alkyne 12 (0.72 g, 99% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 6.99-6.91 (m, 1H), 5.88 (dd, J = 15.6, 1.4 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 2.54 (q, J = 7.2 Hz, 1H), 2.42 (q, J = 7.3 Hz, 1H), 2.00 (s, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.88 (s, 9H), 0.07 (d, J = 6.4 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₆ H ₂₅ D ₃ O ₃ SiNa 322.1894, found 322.1939.

(2-3) Synthesis of _d3 -alkyne 13

Under an argon atmosphere, _d3 -alkyne 13 (0.72 g, 2.41 mmol) was dissolved in dehydrated toluene (24.1 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (7.22 mL, 7.22 mmol) was added dropwise under ice cooling and stirred for 0.5 hours. After stirring, isopropanol (5.05 mL), silica gel, and distilled water (24.1 mL) were added to the reaction solution, stirred, filtered through Celite, and the solvent was removed. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 10/1) to obtain _d3 -alkyne 13 (0.60 g, 98% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.11 (d, J = 4.1 Hz, 2H), 2.41-2.37 (m, 1H), 2.28 (dd, J = 12.6, 4.4 Hz, 1H), 1.98 (s, 1H), 0.89 (s, 9H), 0.07 (d, J = 6.9 Hz, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₂₃ D ₃ O ₂ SiNa 280.1788, found 280.1791.

(2-4) Synthesis of _d3 -alkyne 14
Under an argon atmosphere, methylene chloride (18 mL) was added to MS4A (1.18 g), and a methylene chloride solution (6 mL) of Ti(O ⁱ Pr) ₄ (0.21 mL, 0.70 mmol), L-(+)-DET (0.15 mL, 0.85 mmol), and d ₃ -alkyne 13 (0.60 g, 2.35 mmol) was added at -20°C, and TBHP (3.5 M in CH ₂ Cl ₂ , 1.34 mL, 4.7 mmol) was added dropwise, and the mixture was stirred at the same temperature for 1 hour. After the stirring, dimethyl sulfide (2.5 mL) and a saturated aqueous sodium fluoride solution (70 mL) were added to the reaction solution, and the mixture was stirred for 12 hours. After the stirring, the reaction solution was filtered through Celite (methylene chloride), and the solvent was distilled off. The residue was extracted with methylene chloride, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=20/1) to give d ₃ -alkyne 14 (0.58 g, yield 91%) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 3.89 (dd, J = 12.8, 2.3 Hz, 1H), 3.57 (dd, J = 12.6, 4.4 Hz, 1H), 3.08-3.05 (m, 1H), 2.96-2.94 (m, 1H), 1.97 (s, 1H), 1.83-1.80 (m, 1H), 1.74 (q, J = 7.2 Hz, 1H), 0.87 (s, 9H), 0.07 (s, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₂₃ D ₃ O ₃ SiNa 296.1737, found 296.1736.

(2-5) Synthesis of _d3 -alkyne 15
Under an argon atmosphere, d ₃ -alkyne 14 (0.51 g, 1.85 mmol) was dissolved in anhydrous tetrahydrofuran solution (9.25 mL), and triphenylphosphine (1.46 g, 5.55 mmol), imidazole (756 mg, 11.1 mmol), and iodine (1.41 g, 5.55 mmol) were added in sequence at −20° C., and the mixture was stirred at the same temperature for 0.5 hours, and then stirred at room temperature for 0.5 hours. After the stirring, a saturated aqueous solution of sodium thiosulfate (9.25 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 80/1) to obtain d ₃ -alkyne 15 (0.62 g, 88% yield) as a colorless liquid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 3.24-3.19 (m, 1H), 3.13-3.00 (m, 2H), 2.99-2.95 (m, 1H), 1.98 (s, 1H), 1.90-1.82 (m, 1H), 1.74-1.66 (m, 1H), 0.89 (s, 9H), 0.09 (s, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₂₂ D ₃ IO ₂ SiNa 406.0755, found 406.0762.

(2-6) Synthesis of _d3 -alkyne 16
_d3 -Alkyne 15 (0.58 g, 1.51 mmol) was dissolved in methanol (14.5 mL), acetic acid (0.58 mL) and zinc metal treated with hydrochloric acid (346 mg, 5.29 mmol) were added, stirred for 1 hour, filtered through Celite, and the solvent was removed. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 80/1) to give _d3 -alkyne 16 (0.41 g, 99% yield) as a colorless liquid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 5.92-5.82 (m, 1H), 5.30-5.24 (m, 1H), 5.10 (qt, J = 5.0, 1.4 Hz, 1H), 4.42-4.38 (m, 1H), 1.99 (s, 1H), 1.93-1.73 (m, 2H), 0.90 (s, 9H), 0.11 (s, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₂₃ D ₃ O ₂ SiNa 280.1788, found 280.1804.

(2-7) Optical resolution of d ₃ -alkyne 16
Under an argon atmosphere, _d3 -alkyne 16 (333 mg, 1.29 mmol) was dissolved in dehydrated toluene (26 mL), and (2S,3R)-HyperBTM (40.1 mg, 0.13 mmol, 10 mol%), _iPr2NEt (68μL), and ( ^COiPr ) _2O (65μL) were added at -60℃, and the mixture was stirred at -60℃ for 8 hours. After stirring, distilled water (20 mL) was added to the reaction solution, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 100/1) to obtain _d3 -alkyne (-)-16 (270 mg, 82% yield) as a colorless liquid.

(2-8) Synthesis of _d3 -alkyne 17
Under an argon atmosphere, d ₃ -alkyne (-)-16 (259.1 mg, 1.01 mmol) was dissolved in dimethylformaldehyde (2.0 mL), and imidazole (116.4 mg, 1.71 mmol), tert-butyldimethylsilyl chloride (167 mg, 1.11 mmol), and 4-dimethylaminopyridine (6.8 mg, 0.056 mmol) were added and stirred at room temperature for 1 hour. After stirring, distilled water (10 mL) was added to the reaction solution, extracted with methylene chloride, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 100/1) to obtain d ₃ -alkyne 17 (308.9 mg, 82% yield) as a colorless liquid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 5.87-5.76 (m, 1H), 5.20-5.02 (m, 2H), 4.23-4.19 (m, 1H), 1.97 (s, 1H), 1.91-1.78 (m, 1H), 1.65 (dd, J = 13.8, 4.8 Hz, 1H), 0.89 (s, 18H), 0.09-0.04 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₂₀ H ₃₇ D ₃ O ₃ Si ₂ Na 394.2653, found 394.2677.

(3) Synthesis of alkyne compound 30

Alkyne compound 30 was prepared by the synthetic route shown below, with each reaction in the synthetic route being explained below.

(3-1) Synthesis of _d5 -triol 19
Triol 18 (1.62 g, 15.3 mmol) was dissolved in deuterium oxide (30 mL) under a hydrogen atmosphere, and ruthenium carbon (5%, 1.62 g, 16.0 mmol) was added at room temperature and stirred at 80° C. for 72 hours. After stirring, the reaction solution was filtered through Celite (ethyl acetate/methanol = 8/1) and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (ethyl acetate/methanol = 8/1) to obtain d ₅ -triol 19 (1.62 g, 100% yield) as a colorless liquid.
^1H NMR (300 MHz, _D2O ) δ 3.13 (s, 1H), 1.45 (dd, J = 31.0, 14.1 Hz, 2H).
HRMS (ESI, M+Na) calcd. for C ₄ H ₅ D ₅ O ₃ Na 134.0842, found 134.0823.

(3-2) Synthesis of d ₅ -alcohol 20
Under an argon atmosphere, _d3 -triol 19 (500 mg, 4.50 mmol) was dissolved in acetone (7 mL), p-toluenesulfonic acid (55.6 mg, 0.292 mmol) was added, and the mixture was stirred at room temperature for 12 hours. After stirring, triethylamine (0.233 mL) was added to the reaction solution, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain _d5 -alcohol 20 (538 mg, 79% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 2.10 (s, 1H), 1.81 (s, 2H), 1.43 (s, 3H), 1.36 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₇ H ₉ D ₅ O ₅ Na 174.1155, found 174.1156.

(3-3) Synthesis of _d5 -ester 21
Under an argon atmosphere, d ₅ -alcohol 20 (538 mg, 3.56 mmol) was dissolved in methylene chloride (7.1 mL), triethylamine (1.49 mL, 10.7 mmol) and benzoyl chloride (0.615 mL, 5.34 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After stirring, distilled water (7 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4/1) to obtain d ₅ -ester 21 (909 mg, 100% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.04-8.01 (m, 2H), 7.58-7.53 (m, 1H), 7.45-7.41 (m, 2H), 2.08-1.96 (m, 2H), 1.42 (s, 3H), 1.36 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₁₄ H ₁₃ D ₅ O ₄ Na 278.1417, found 278.1400.

(3-4) Synthesis of _d5 -diol 22
Ester 21 (909 mg, 3.56 mmol) was dissolved in distilled water (1.34 mL), acetic acid (8.04 mL) was added at room temperature, and the mixture was stirred for 12 hours, after which the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain _d5 -diol 22 (784 mg, 88% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.03-8.00 (m, 2H), 7.58-7.52 (m, 1H), 7.45-7.40 (m, 2H), 2.73 (s, 2H), 1.85 (dd, J = 23.4, 14.1 Hz, 2H).
HRMS (ESI, M+Na) calcd. for C ₁₁ H ₉ D ₅ O ₄ Na 238.1104, found 238.1125.

(3-5) Synthesis of _d5 -bissilyl ether 23
Under an argon atmosphere, d ₅ -triol 22 (784 mg, 3.64 mmol) was dissolved in methylene chloride (18 mL), and 2,6-lutidine (1.12 mL, 9.47 mmol) and tert-butyldimethylsilyl triflate (2.09 ml, 9.11 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. After stirring, distilled water (18 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 20/1) to obtain d ₅ -bissilyl ether 23 (1.77 g, 100% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.06-8.03 (m, 2H), 7.58-7.53 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H), 2.07 (d, J = 14.1 Hz, 1H), 1.82-1.75 (m, 1H), 0.89 (s, 18H), 0.06 (t, J = 2.4 Hz, 12H).
HRMS (ESI, M+Na) calcd. for C ₂₃ H ₃₇ D ₅ O ₄ Si ₂ Na 466.2833, found 466.2862.

(3-6) Synthesis of _d5 -alcohol 24

Under an argon atmosphere, d ₅ -bissilyl ether 23 (195 mg, 0.438 mmol) was dissolved in methanol (2 mL) and dichloromethane (2 mL), camphorsulfonic acid (5.1 mg, 0.0219 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After stirring, saturated aqueous sodium bicarbonate solution (4 mL) was added to the reaction mixture, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate) to obtain d ₅ -alcohol 24 (77 mg, 58% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.04-8.01 (m, 2H), 7.59-7.54 (m, 1H), 7.44 (t, J = 7.7 Hz, 2H), 2.02-1.76 (m, 2H), 0.94-0.89 (m, 9H), 0.12-0.07 (m, 6H).
HRMS (ESI, M+Na) calcd. for C ₁₇ H ₂₃ D ₅ O ₄ SiNa 352.1968, found 352.2010.

(3-7) Synthesis of _d4 -alkyne 25
Under an argon atmosphere, oxalyl chloride (0.24 mL, 2.8 mmol) was dissolved in dehydrated methylene chloride (7 mL), and dimethyl sulfoxide (0.497 mL, 7.00 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 10 minutes, and then a methylene chloride solution (7 mL) of d ₅ -alcohol 24 (461 mg, 1.40 mmol) and triethylamine (1.94 mL, 14.0 mmol) were added dropwise in sequence. The reaction mixture was stirred at the same temperature for another hour. After the stirring, distilled water (14 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue (containing d ₄ -aldehyde) was used in the next step without further purification.
Under an argon atmosphere, trimethylsilylacetylene (0.996 mL, 7.2 mmol) was dissolved in anhydrous tetrahydrofuran solution (12 mL), and n-butyllithium (2.6 M hexane solution) (2.3 mL, 6.0 mmol) was added dropwise at -78°C to prepare a reaction mixture. After stirring the reaction mixture at the same temperature for 30 minutes, a tetrahydrofuran solution (12 mL) of d ₄ -aldehyde (772 mg, 2.40 mmol) was added dropwise in sequence. After the addition, the reaction mixture was stirred at the same temperature for another 15 minutes. After the stirring, a saturated aqueous ammonium chloride solution (24 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 30/1) to obtain d ₄ -alkyne 25 (910 mg, 90% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.06-8.02 (m, 2H), 7.59-7.54 (m, 1H), 7.45 (t, J = 7.6 Hz, 2H), 2.38-1.89 (m, 2H), 0.93-0.90 (m, 9H), 0.16-0.06 (m, 15H).
HRMS (ESI, M+Na) calcd. for C ₂₂ H ₃₂ D ₄ O ₄ Si ₂ Na 447.2301, found 447.2289.

(3-8) Synthesis of _d4 -alkyne 26
Under an argon atmosphere, d ₄ -alkyne 25 (297 mg, 0.700 mmol) was dissolved in methylene chloride (7 mL), and 2,6-lutidine (0.199 mL, 1.68 mmol) and tert-butyldimethylsilyl triflate (0.193 ml, 0.840 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After stirring, distilled water (7 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 40/1) to obtain d ₄ -bissilyl ether 26 (381 mg, 100% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.06-8.03 (m, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 2.19-1.90 (m, 2H), 0.93-0.87 (m, 18H), 0.24-0.03 (m, 21H).
HRMS (ESI, M+Na) calcd. for C ₂₈ H ₄₆ D ₄ O ₄ Si ₃ Na 561.3166, found 561.3141.

(3-9) Synthesis of _d4 -alkyne 27
D ₄ -Alkyne 26 (314 mg, 0.587 mmol) was dissolved in methanol (2 mL), and potassium carbonate (324 mg, 2.35 mmol) was added under ice cooling, followed by stirring at room temperature for 1 hour. After stirring, distilled water (2 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=30/1) to obtain d ₄ -alcohol 27 (152 mg, 76% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 2.41 (s, 1H), 1.99-1.59 (m, 2H), 0.90 (d, J = 1.7 Hz, 18H), 0.16-0.09 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₈ H ₃₄ D ₄ O ₃ Si ₂ Na 385.2508, found 385.2549.

(3-10) Synthesis of _d4 -alkyne 28
Under an argon atmosphere, d ₄ -alcohol 27 (136 mg, 0.375 mmol) was dissolved in anhydrous tetrahydrofuran solution (1.6 mL), and triphenylphosphine (509 mg, 2.40 mmol), imidazole (76.6 mg, 1.13 mmol), and iodine (306 mg, 1.20 mmol) were added in sequence at -20°C and stirred for 30 minutes, and then stirred at room temperature for 20 minutes. After stirring, saturated sodium thiosulfate solution (1.6 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane) to obtain d ₄ -iodide 28 (174 mg, 98% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 2.39 (s, 1H), 2.20-2.06 (m, 2H), 0.90 (d, J = 2.1 Hz, 18H), 0.14-0.12 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₈ H ₃₃ D ₄ IO ₂ Si ₂ Na 495.1526, found 495.1497.

(3-11) Synthesis of _d4 -nitrile 29
Under an argon atmosphere, _d4 -iodide 28 (174 mg, 0.369 mmol) was dissolved in dimethylformamide (0.9 mL), sodium cyanide (27.1 mg, 0.554 mmol) was added at room temperature, and the mixture was stirred at 90°C for 20 minutes. After stirring, saturated aqueous sodium bicarbonate solution (1 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 80/1) to obtain _d4 -nitrile 29 (130 mg, 94% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 2.42 (s, 1H), 1.98 (dd, J = 24.1, 13.8 Hz, 2H), 0.90 (s, 18H), 0.14-0.11 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₉ H ₃₃ D ₄ NO ₂ Si ₂ Na 394.2540, found 394.2512.

(3-12) Synthesis of _d4 -alkyne 30

Under an argon atmosphere, _d4 -nitrile 29 (108 mg, 0.291 mmol) was dissolved in dehydrated dichloromethane (1.5 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (0.35 mL, 0.35 mmol) was added dropwise under ice cooling and stirred for 20 minutes. After stirring, isopropanol (0.245 mL), silica gel (700 mg), and distilled water (1 mL) were added to the reaction solution, and the mixture was stirred for 30 minutes. After filtration through Celite, the solvent was distilled off. After distillation of the solvent, the residue (containing _d4 -aldehyde) was used in the next step without further purification.
Under an argon atmosphere, methyltriphenylphosphonium iodide (623 mg, 1.54 mmol) was dissolved in anhydrous tetrahydrofuran solution (1.5 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (0.766 mL, 1.46 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then a tetrahydrofuran solution (1.5 mL) of d ₄ -aldehyde (109 mg, 0.291 mmol) was added dropwise to prepare a reaction mixture. The reaction mixture was stirred at the same temperature for another 30 minutes. After the stirring, a saturated aqueous ammonium chloride solution (3 mL) was added to the reaction mixture, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 100/0) to obtain d ₄ -alkyne 30 (55.9 mg, 52% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 5.88-5.77 (m, 1H), 5.05-4.94 (m, 2H), 2.35 (s, 1H), 1.78-1.56 (m, 2H), 0.91 (s, 18H), 0.12 (dd, J = 13.8, 7.9Hz, 12H).
HRMS (ESI, M+Na) calcd. for C ₂₀ H ₃₆ D ₄ O ₂ Si ₂ Na 395.2716, found 395.2699.

(4) Synthesis of non-isotopically labeled alkyne compound 42

In the experiments described below, a non-isotopically labeled vitamin D compound was used. To produce the vitamin D compound, a non-isotopically labeled alkyne compound 42 was prepared by the synthetic route shown below. Each reaction in the synthetic route is described below.

(4-1) Synthesis of alcohol 32

Triol 31 (4.0 g, 37.3 mmol) was dissolved in acetone (60 mL) under an argon atmosphere, and p-toluenesulfonic acid (461 mg, 2.4 mmol) was added and stirred at room temperature for 12 hours. Triethylamine (1.9 mL) was added to the reaction solution, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain alcohol 32 (2.8 g, 52% yield) as a colorless liquid.

(4-2) Synthesis of ester 33

Under an argon atmosphere, alcohol 32 (2.8 g, 3.6 mmol) was dissolved in methylene chloride (40 mL), triethylamine (8 mL, 58.0 mmol) and benzoyl chloride (3.3 mL, 29.0 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After stirring, distilled water (40 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4/1) to obtain ester 33 (4.8 g, 100% yield) as a colorless liquid.

(4-3) Synthesis of diol 34

Ester 33 (5.6 g, 22.6 mmol) was dissolved in distilled water (6.5 mL), acetic acid (40 mL) was added at room temperature, and the mixture was stirred for 12 hours, after which the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain diol 34 (4.4 g, 88% yield) as a colorless liquid.

(4-4) Synthesis of bissilyl ether 35

Under an argon atmosphere, diol 34 (300 mg, 1.4 mmol) was dissolved in methylene chloride (7.0 mL), and 2,6-lutidine (0.44 mL, 3.7 mmol) and tert-butyldimethylsilyl triflate (0.82 ml, 3.6 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. After stirring, distilled water (7.0 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=20/1) to obtain bissilyl ether 35 (627 mg, 100% yield) as a colorless liquid.

(4-5) Synthesis of alcohol 36

Under an argon atmosphere, bissilyl ether 35 (627 mg, 1.4 mmol) was dissolved in methanol (8.5 mL) and dichloromethane (8.5 mL), camphorsulfonic acid (40 mg, 0.17 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After stirring, saturated aqueous sodium bicarbonate solution (17 mL) was added to the reaction solution, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=10/1) to obtain alcohol 36 (77 mg, 54% yield) as a colorless liquid.

(4-6) Synthesis of alkyne 37

Under an argon atmosphere, oxalyl chloride (0.48 mL, 5.6 mmol) was dissolved in dehydrated methylene chloride (7.0 mL), and dimethyl sulfoxide (1.0 mL, 14.0 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 10 minutes, and then a methylene chloride solution (7.0 mL) of alcohol 36 (902 mg, 2.78 mmol) and triethylamine (3.9 mL, 27.8 mmol) were added dropwise in sequence. The reaction mixture was stirred at the same temperature for another hour. After the stirring, distilled water (14 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was filtered using silica gel column chromatography (hexane/ethyl acetate = 1/1) to obtain the aldehyde. This was used in the next step without further purification.
Under an argon atmosphere, trimethylsilylacetylene (1.0 mL, 7.2 mmol) was dissolved in anhydrous tetrahydrofuran solution (12 mL), and n-butyllithium (2.6 M hexane solution) (2.3 mL, 6.0 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 30 minutes, and then the tetrahydrofuran solution of the aldehyde (12 mL) was added dropwise in sequence. The reaction mixture was stirred at the same temperature for another 15 minutes. After the stirring, a saturated aqueous ammonium chloride solution (24 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 30/1) to obtain alkyne 37 (910 mg, 90% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.05-8.03 (m, 2H), 7.59-7.53 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H), 4.56-4.26 (m, 3H), 4.04-3.99 (m, 1H), 2.33-1.87 (m, 2H), 0.92-0.88 (m, 9H), 0.16-0.08 (m, 15H).
HRMS (ESI, M+Na) calcd. for C ₂₂ H ₃₆ O ₄ Si ₂ Na 443.2050, found 443.2017.

(4-7) Synthesis of alkyne 38

Under an argon atmosphere, alkyne 37 (264 mg, 0.63 mmol) was dissolved in methylene chloride (6.0 mL), and 2,6-lutidine (0.22 mL, 1.9 mmol) and tert-butyldimethylsilyl triflate (0.22 ml, 0.94 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After stirring, distilled water (6.0 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 40/1) to obtain alkyne 38 (334 mg, 100% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.06-8.03 (m, 2H), 7.58-7.53 (m, 1H), 7.44 (t, J = 7.4 Hz, 2H), 4.54-4.36 (m, 2H), 4.28 (d, J = 4.5 Hz, 1H), 3.94-3.89 (m, 1H), 2.19-1.92 (m, 2H), 0.91-0.87 (m, 18H), 0.15-0.02 (m, 21H).
HRMS (ESI, M+Na) calcd. for C ₂₈ H ₅₀ O ₄ Si ₃ Na 557.2915, found 557.2872.

(4-8) Synthesis of alkyne 39

Alkyne 38 (206 mg, 0.39 mmol) was dissolved in methanol (1.3 mL), and potassium carbonate (213 mg, 1.5 mmol) was added under ice cooling, followed by stirring at room temperature for 1 hour. After stirring, distilled water (1.3 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 10/1) to obtain alkyne 39 (104 mg, 76% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.36 (q, J = 2.2 Hz, 1H), 3.94 (q, J = 4.8 Hz, 1H), 3.80-3.75 (m, 2H), 2.41 (d, J = 2.1 Hz, 1H), 2.15-1.82 (m, 3H), 0.90 (d, J = 1.7 Hz, 18H), 0.16-0.09 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₈ H ₃₈ O ₃ Si ₂ Na 381.2257, found 381.2263.

(4-9) Synthesis of iodide 40

Under an argon atmosphere, alkyne 39 (136 mg, 0.38 mmol) was dissolved in anhydrous tetrahydrofuran solution (1.6 mL), and triphenylphosphine (193 mg, 0.91 mmol), imidazole (76.6 mg, 1.1 mmol), and iodine (306 mg, 1.2 mmol) were added in sequence at -20°C, and the mixture was stirred for 30 minutes, and then stirred at room temperature for 20 minutes. After the stirring, saturated sodium thiosulfate solution (1.6 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 100/0) to obtain iodide 40 (149 mg, 84% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.25 (q, J = 2.1 Hz, 1H), 3.79-3.68 (m, 1H), 3.41-3.18 (m, 2H), 2.39 (d, J = 2.4 Hz, 1H), 2.19-2.12 (m, 2H), 0.90 (d, J = 2.1 Hz, 18H), 0.14-0.12 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₈ H ₃₇ IO ₂ Si ₂ Na 491.1274, found 491.1243.

(4-10) Synthesis of nitrile 41

Under an argon atmosphere, iodide 40 (133 mg, 0.28 mmol) was dissolved in dimethylformamide (0.7 mL), sodium cyanide (21.0 mg, 0.43 mmol) was added at room temperature, and the mixture was stirred at 90°C for 20 minutes. After stirring, saturated aqueous sodium bicarbonate solution (1.0 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 80/1) to obtain nitrile 41 (105 mg, 94% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 4.27 (q, J = 2.1 Hz, 1H), 3.85-3.80 (m, 1H), 2.51-2.36 (m, 3H), 2.12-1.89 (m, 2H), 0.90 (s, 18H), 0.15-0.11 (m, 12H).
HRMS (ESI, M+Na) calcd. for C ₁₉ H ₃₇ NO ₂ Si ₂ Na 390.2261, found 390.2249.

(4-11) Synthesis of alkyne 42

Under an argon atmosphere, nitrile 41 (32.5 mg, 0.089 mmol) was dissolved in dehydrated dichloromethane (0.4 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (0.11 mL, 0.11 mmol) was added dropwise under ice cooling, and the mixture was stirred for 20 minutes. After the stirring, isopropanol (0.074 mL), silica gel (200 mg), and distilled water (1 mL) were added to the reaction solution, and the mixture was stirred for 30 minutes. After filtration through Celite, the solvent was distilled off. After the solvent was distilled off, the residue was filtered using silica gel column chromatography (hexane/ethyl acetate = 10/1) to obtain the aldehyde. This was used in the next step without further purification.
Under an argon atmosphere, methyltriphenylphosphonium iodide (623 mg, 1.5 mmol) was dissolved in anhydrous tetrahydrofuran solution (0.75 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (0.77 mL, 1.5 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then a tetrahydrofuran solution (0.75 mL) of the aldehyde (109 mg, 0.29 mmol) was added dropwise. The reaction mixture was stirred at the same temperature for another 30 minutes. After the stirring, a saturated aqueous ammonium chloride solution (3 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=100/0) to obtain alkyne 42 (55.9 mg, 62% yield) as a colorless liquid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 5.89-5.76 (m, 1H), 5.05-4.93 (m, 2H), 4.23 (q, J = 2.3 Hz, 1H), 3.73 (dd, J = 10.8, 4.6 Hz, 1H), 2.35 (d, J = 2.4 Hz, 1H), 2.20-2.07 (m, 2H), 1.79-1.59 (m, 2H), 0.91 (s, 18H), 0.11 (dd, J = 13.4, 7.6 Hz, 12H).
HRMS (ESI, M+Na) calcd. for C ₂₀ H ₄₀ O ₂ Si ₂ Na 391.2465, found 391.2442.

(6) Synthesis of Vitamin D Compound 44 (25OH D ₃ -3,4,4'-d ₃ , also known as "25OHD ₃ -d ₃ ")

As shown in the following reaction scheme, the alkyne compound 10 (d ₃ -alkyne 10) prepared in (1) above was reacted with a bromoolefin 43 to produce a vitamin D compound 44.

Under an argon atmosphere, d ₃ -alkyne 10 (10 mg, 0.04 mmol) and bromoolefin 60 (29.3 mg, 0.06 mmol) were dissolved in dehydrated toluene (1.0 mL) and triethylamine (1.0 mL), and triphenylphosphine tetrakispalladium was added and stirred at 90°C for 1 hour. After stirring, the reaction solution was filtered through Celite (ethyl acetate) and the solvent was distilled off. After distilling off the solvent, the residue (containing vitamin D 3-3, 25-bissilyl ether-d ₃ (VD ₃ -3,25-bissilyl ether-d ₃ ) described below) was filtered using silica gel column chromatography (hexane/ethyl acetate = 50/1). This was used in the next step without further purification.

Under an argon atmosphere, _VD3-3,25 -bissilyl ether- _d3 was dissolved in methanol (0.5 mL), and methanesulfonic acid (10 μL) was added dropwise under ice cooling and stirred for 30 minutes. After stirring, saturated aqueous sodium bicarbonate was added to the reaction solution, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) and reverse phase HPLC to obtain vitamin D compound 44 ( _25OHD3 - _d3 , 0.29 mg, yield 1.8% 2 steps) as a colorless solid.
The physical property data measured for the produced vitamin D compound 44 are as follows, and the NMR spectrum data thereof is shown in Figure 1A.
¹ H NMR (400 MHz, CDCl ₃ ) δ 6.23 (d, J = 11.5 Hz, 1H), 6.03 (d, J = 11.5 Hz, 1H), 5.05 (s, 1H), 4.82 (s, 1H), 2.82 (d, J = 12.6 Hz, 1H), 2.37-2.42 (m, 1H), 2.15-2.20 (m, 1H), 1.96-2.04 (m, 2H), 1.84-1.94 (m, 2H), 1.25-1.70 (m, 16H), 1.21 (s, 6H), 0.95 (d, J = 5.2 Hz, 3H), 0.54 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₁ D ₃ O ₂ Na 426.3427, found 426.3454.

(7) Synthesis of Vitamin D Compound 46 (25OHD ₃ -3,4,4'-d ₃ -26R,23S-lactone, also known as "25OHD ₃ -26R,23S-lactone-d ₃ ")

Vitamin D compound 46 was synthesized by carrying out the same synthesis as in (6) above, except that the following bromoolefin 45 was used instead of bromoolefin 60.

The physical property data measured for the produced vitamin D compound 46 are as follows, and the NMR spectrum data thereof is shown in FIG.
¹ H NMR (400 MHz, CDCl ₃ ) δ 6.23 (d, J = 11.4 Hz, 1H), 6.03 (d, J = 10.5 Hz, 1H), 5.05 (s, 1H), 4.81 (s, 1H), 4.54-4.36 (m, 1H), 3.75-3.56 (m, 2H), 2.83 (d, J = 12.8 Hz, 1H), 2.51-2.31 (m, 2H), 2.26-2.10 (m, 1H), 2.11-1.80 (m, 6H), 1.81-1.43 (m, 8H), 1.42-1.15 (m, 6H), 1.02 (d, J = 6.4 Hz, 0.55 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₃₇ D ₃ O ₄ Na 454.3013, found 454.3009.

(8) Synthesis of vitamin D compound 47 (1α,25(OH) ₂ D ₃ -3,4,4'-d ₃ , also known as "1α,25(OH) ₂ D ₃ -d ₃ ")

Vitamin D compound 47 was synthesized by carrying out the same synthesis as in (6) above, except that alkyne compound 17 was used instead of alkyne compound 10.

The physical property data measured for the produced vitamin D compound 47 are as follows, and the NMR spectrum data thereof is shown in FIG. 1C.
¹ H NMR (300 MHz, CDCl ₃ ) δ 6.38 (d, J = 11.0 Hz, 1H), 6.01 (d, J = 11.7 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.42-4.42 (m, 1H), 2.82 (d, J = 12.4 Hz, 1H), 1.25-2.05 (m, 20H), 1.21 (s, 6H), 0.93 (d, J = 6.2 Hz, 3H), 0.54 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₁ D ₃ O ₃ Na 442.3376, found 442.3336.

(9) Synthesis of vitamin D compound 48 (1α,25(OH) ₂ D ₃ -3,4,4'-d ₃ -26R,23S-lactone, also known as "1α,25(OH) ₂ D ₃ -26R,23S-lactone-d ₃ ")

The following vitamin D compound 48 was synthesized by carrying out the same synthesis as in (6) above, except that the above bromoolefin 45 was used instead of bromoolefin 43 and the alkyne compound 17 was used instead of alkyne compound 10.

The physical property data measured for the prepared vitamin D compound 48 are as follows, and the NMR spectrum data thereof is shown in FIG.
¹ H NMR (300 MHz, CDCl ₃ ) δ 6.37 (d, J = 11.4 Hz, 1H), 6.01 (d, J = 10.7 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.46-4.46 (m, 2H), 2.83 (d, J = 13.1 Hz, 1H), 2.39 (dd, J = 13.2, 5.0 Hz, 1H), 1.25-2.05 (m, 20H), 1.02 (d, J = 6.5 Hz, 3H), 0.55 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₃₇ D ₃ O ₅ Na 470.2962, found 470.2963.

(10) Synthesis of vitamin D compound 49 (24R,25(OH) _2D3-3,4,4' - _d3 , also known as "24R,25(OH) _2D3 - _{d3 "} ) _.

The following vitamin D compound 50 was synthesized by carrying out the same synthesis as in (6) above, except that the following bromoolefin 49 was used instead of the bromoolefin 43.

The physical property data measured for the produced vitamin D compound 67 are as follows, and the NMR spectrum data thereof is shown in FIG. 1E.
¹ H NMR (400 MHz, CDCl ₃ ) δ 6.23 (d, J = 11.0 Hz, 1H), 6.03 (d, J = 11.4 Hz, 1H), 5.05 (s, 1H), 4.82 (s, 1H), 3.34 (t, J = 6.0 Hz, 1H), 2.83 (d, J = 12.8 Hz, 1H), 2.35-2.42 (m, 1H), 2.18-2.20 (m, 1H), 1.16-2.01 (m, 24H), 0.94 (d, J = 6.4 Hz, 3H), 0.55 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₁ D ₃ O ₃ Na 442.3376, found 442.3402.

(11) Synthesis of vitamin D compound 51 (1α,24R,25(OH) ₃ -3,4,4'-d ₃ D ₃ , also known as "1α,24R,25(OH) ₃ D ₃ -d _{3 "} )

The following vitamin D compound 51 was synthesized by carrying out the same synthesis as in (6) above, except that the above-mentioned bromoolefin 49 was used instead of bromoolefin 43 and the alkyne compound 17 was used instead of alkyne compound 10.

The physical property data measured for the prepared vitamin D compound 51 are as follows, and the NMR spectrum data thereof is shown in FIG.
¹ H NMR (500 MHz, CD ₃ OD) δ 6.36 (d, J = 11.5 Hz, 1H), 6.12 (d, J = 11.5 Hz, 1H), 5.33 (s, 1H), 4.90 (s, 1H), 4.38 (t, J = 6.0 Hz, 1H), 3.24 (d, J = 9.7 Hz, 1H), 2.87-2.92 (m, 1H), 1.24-2.09 (m, 18H), 1.20 (s, 3H), 1.16 (s, 3H), 1.00 (d, J = 6.3 Hz, 3H), 0.62 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₁ D ₃ O ₄ Na 458.3326, found 458.3334.

(12) Vitamin D compound 52 (4β,25(OH) _2D3-1,1 ',3,4- _d3 , also known as "4β,25(OH _{) 2D3 - d4} ")

Vitamin D compound 52 was synthesized by carrying out the same synthesis as in (6) above, except that alkyne compound 30 was used instead of alkyne compound 10.

Under an argon atmosphere, alkyne compound 30 ( _d4 -alkyne, 6.5 mg, 0.027 mmol) and bromoolefin 43 (9.8 mg, 0.032 mmol) were dissolved in dehydrated toluene (0.3 mL) and triethylamine (0.3 mL), and triphenylphosphinetetrakispalladium was added and stirred at 90°C for 1 hour. After stirring, the reaction solution was filtered through Celite (ethyl acetate) and the solvent was distilled off. After distilling off the solvent, the residue (containing vitamin _D3-3,4,25 -trisilyl ether- _d4 ( _VD3-3,4,25 -trisilyl ether- _d4 ) described below) was filtered using silica gel column chromatography (hexane/ethyl acetate = 50/1). This was used in the next step without further purification.

Under an argon atmosphere, VD ₃ -3,4,25-trisilyl ether-d ₄ was dissolved in tetrahydrofuran solution (0.4 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution) (0.075 mL, 0.075 mmol) was added dropwise under ice cooling and stirred for 12 hours. After stirring, a saturated aqueous ammonium chloride solution was added to the reaction solution, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) and reverse phase HPLC to obtain vitamin D compound 52 (d4-4β,25(OH) ₂ vitamin D ₃ , 1.8 mg, yield 27% 2 steps) as a colorless solid.
The physical property data measured for the prepared vitamin D compound 52 are as follows, and the NMR spectrum data thereof is shown in FIG. 1G.
¹ H NMR (400 MHz, CDCl ₃ ) δ 6.51 (d, J = 11.4 Hz, 1H), 6.04 (d, J = 11.4 Hz, 1H), 5.17 (d, J = 2.3 Hz, 1H), 4.92 (d, J = 2.3 Hz, 1H), 2.86 (d, J = 13.7 Hz, 1H), 2.00-1.98 (m, 2H), 1.90-1.80 (m, 2H), 1.53-1.34 (m, 5H), 1.32-1.25 (m, 1H), 1.22 (s, 6H), 1.09-1.04 (m, 1H), 0.94 (d, J = 6.4 Hz, 3H), 0.54 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₀ D ₄ O ₃ Na 443.3439, found 443.3459.

(13) Synthesis _of non-isotopically labeled vitamin D compound 54 (4β,25(OH) _2D3 )

Vitamin D compound 54 shown below was synthesized by carrying out the same synthesis as in (12) above, except that non-isotopically labeled alkyne compound 53 was used instead of alkyne compound 30.

The physical property data of the obtained vitamin D compound 54 are as follows, and the NMR spectrum data thereof is shown in FIG.
¹ H NMR (500 MHz, CDCl ₃ ) δ 6.51 (d, J = 10.9 Hz, 1H), 6.04 (d, J = 11.5 Hz, 1H), 5.17 (s, 1H), 4.92 (s, 1H), 4.22 (s, 1H), 3.86 (s, 1H), 2.86 (d, J = 13.2 Hz, 1H), 2.38-2.35 (m, 1H), 2.18-2.13 (m, 1H), 2.01 (d, J = 10.9 Hz, 2H), 1.85 (d, J = 6.3 Hz, 3H), 1.71-1.33 (m, 17H), 1.21 (s, 6H), 1.05 (q, J = 9.9 Hz, 1H), 0.93 (d, J = 6.3 Hz, 3H), 0.54 (s, 3H).
HRMS (ESI, M+Na) calcd. for C ₂₇ H ₄₀ D ₄ O ₃ Na 439.3188, found 439.3181.

6-2. Example 2: LC-MS/MS analysis of 25OHD ₃ -26R,23S-lactone and 1α,25(OH) ₂ D ₃ -26R,23S-lactone in pooled serum

The stable isotope-labeled vitamin D compounds synthesized in Example 1 were used to quantitatively analyze the two types of vitamin D compounds contained in pooled serum by LC-MS/MS. The procedure for this quantitative analysis is as follows.

(1) Preparation of calibrator
30% acetonitrile solutions of 25OHD ₃ -26R,23S-lactone standard and 1α,25(OH) ₂ D ₃ -26R,23S-lactone standard were prepared at the concentrations shown in Table 2 below.

(2) Preparation of IS (internal standard) solution ₃₀ % acetonitrile solutions of authentic vitamin D compound 46 ( _25OHD3-26R ,23S-lactone- _d3 ) and vitamin D compound 48 (1α,25(OH) _2D3-26R ,23S-lactone- _d3 ) were prepared at the concentrations shown in Table 3 below.

(3) Sample pretreatment Sample pretreatment was carried out according to the following procedure.
(3-1) SLE (solid-liquid) extraction
1) 100 μL of serum and 200 μL of IS solution were mixed.
2) 300 μL of serum/IS mixture was loaded onto the SLE column.
3) 600 μL of hexane/ethyl acetate (50/50, v/v) was added for elution.
This was repeated three times.
(3-2) Drying: Nitrogen spray or centrifugal concentration for about 20 minutes
(3-3) Derivatization
1) Preheat the derivatization reagent DAP-PA (80°C, 15 minutes). (The structure of DAP-PA and its derivatization reaction are shown in Figure 2G.)
2) 100 μL of DAP-PA (pre-warmed) was added to the dried sample.
3) Leave to stand at room temperature (15 minutes).
4) Drying: Centrifugal concentration for about 15 minutes
5) Dissolve in 50 μL of reconstitution solution (50% acetonitrile).

(4) LC analysis conditions
The LC analysis conditions were as follows.
Equipment: Waters ACQUITY UPLC I-Class
Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 75 mm
Elution conditions: flow rate 0.4 mL/min. Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile. Details of the elution conditions are as shown in Table 4 below.

(5) MS/MS analysis conditions
The MS/MS analysis conditions were as follows:
Apparatus: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode
The SRM parameters were as shown in Table 5 below.

(6) Analysis Results of the Specimens The analysis results of the specimens are shown in Figs. 2A to 2D.
FIG. 2A is an SRM chromatogram of a 25OHD ₃ -26R,23S-lactone-d ₃ preparation.
FIG. 2B is an SRM chromatogram of a 1α,25(OH) ₂ D ₃ -26R,23S-lactone-d ₃ preparation.
FIG. 2C is a calibration curve for the 25OHD ₃ -26R,23S-lactone standard.
FIG. 2D is a calibration curve for the 1α,25(OH) ₂ D ₃ -26R,23S-lactone standard.

(7) Quantitative Values of _25OHD3-26R ,23S-lactone and 1α,25(OH) _2D3-26R ,23S-lactone in Pooled Serum Based on the above analytical results, _{the amounts of 25OHD3-26R,23S-lactone and 1α,25(OH)2D3-26R , 23S -} lactone in pooled serum were quantified as shown in Table 6. Thus, by using the vitamin D compound of the present invention as a standard substance (particularly an internal standard substance) in mass spectrometry, vitamin D metabolites in a sample (particularly a biological sample) can be quantified.

FIG. 2E is an SRM chromatogram of 25OHD ₃ -26R,23S-lactone in pooled serum.
FIG. 2F is an SRM chromatogram of 1α,25(OH) ₂ D ₃ -26R,23S-lactone in pooled serum.

6-3. Example 3: LC-MS/MS analysis of 25OHD3 _and 24R, ₂₅ (OH) _2D3 in pooled serum

The stable isotope-labeled vitamin D compounds synthesized in Example 1 were used to quantitatively analyze the two types of vitamin D compounds contained in pooled serum by LC-MS/MS. The procedure for the quantitative analysis was as follows.

(1) Calibrator JeoQuant Kit for LC/MS/MS Analysis of Vitamin D Metabolites (JEOL Ltd.) was used as the calibrator. The concentrations were as shown in Table 7 below.

(2) IS Solution 30% acetonitrile solutions of authentic vitamin D compound 44 (25OHD ₃ -d ₃ ) and vitamin D compound 50 (24R,25(OH) ₂ D ₃ -d ₃ ) were prepared at the concentrations shown in Table 8 below.

(3) Sample pretreatment Sample pretreatment was carried out according to the following procedure.
(3-1) SLE (solid-liquid) extraction
1) Mix 50 μL of serum and IS solution (20 μL of 25OHD ₃ -d ₃ , 2 μL of 24R,25(OH) ₂ D ₃ -d ₃ ) and dilute with 230 μL of 30% acetonitrile solution.
2) 302 μL of serum/IS mixture was loaded onto the SLE column.
3) 600 μL of hexane/ethyl acetate (50/50, v/v) was added for elution.
This was repeated twice.
(3-2) Centrifugal concentration for about 20 minutes
(3-3) Derivatization
1) Add 100 μL of the derivatization reagent DAP-PA and heat at 80°C for 15 minutes (see Figure 2H).
2) Drying: Centrifugal concentration for about 15 minutes
3) Dissolve in 100 μL of reconstitution solution (50% acetonitrile).

(4) LC analysis conditions: Shimadzu Corporation UFLC LC-20AD
Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 100 mm
Elution conditions: Flow rate 0.3 mL/min. Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile. Detailed elution conditions were as shown in Table 9 below.

(5) MS/MS analysis conditions: Instrument: Shimadzu Corporation LCMS8040 triple quadrupole mass spectrometer; Ionization conditions: ESI positive ion mode
The SRM parameters were as shown in Table 10 below.

(6) Results of Analysis of the Specimens The results of the analysis of the specimens are shown in Figs. 3A to 3D.
FIG. 3A is a calibration curve for _25OHD3 standard.
FIG. 3B is a calibration curve for the 24R,25(OH) ₂ D ₃ standard.

(7) Quantitative Values _{of 25OHD3 and 24R,25(OH)2D3 in Pooled} Serum Based on the above analytical results, _25OHD3 and 24R,25(OH) _2D3 in pooled serum were quantified as shown in Table 11. Thus, by using the vitamin D compound of the present invention as _a standard substance (particularly an internal standard substance) in mass spectrometry, vitamin D metabolites in a sample (particularly a biological sample) can be quantified.

FIG. 3C is an SRM chromatogram of _25OHD3 in pooled serum.
FIG. 3D is _an SRM chromatogram of 24R,25(OH) _2D3 in pooled serum.

6-4. Example 4: Separation _and measurement of 4β,25-(OH) _2D3

There are two or more vitamin D compounds that have different positions or types of groups bonded to the A ring, but the molecular weights of the substances generated by cleavage after derivatization for mass spectrometry are exactly the same. It has been difficult to detect or quantify each of the two or more vitamin D compounds.
Each of said two or more vitamin D compounds can be detected and/or quantified by an analytical method using the vitamin D compounds of the present invention and carrying out a specific procedure.
Below, an example of quantifying 4β,25-(OH) ₂ D ₃ using this analytical method will be described.

(1) Material standard: 4β,25-(OH) _2D3 ( _vitamin D compound 54 produced in Example 1 above)
Stable isotope-labeled compound: 4β,25-(OH) ₂ D ₃ -d ₄ (vitamin D compound 52 produced in Example 1 above)
Vitamin D-free serum: MSG1000 (Golden West Biologicals)
Antibody column: 1,25-(OH) _2- Vitamin _D3 / _D2 Immuno Tube™ Extraction Kit (Immunodiagnostik)
SLE column: Isolute SLE+ column (Biotage)
Derivatization reagent (DAP-PA): JeoQuant Kit for LC-MS/MS Analysis of Vitamin D Metabolites (JEOL Ltd.)

(2) IS (internal standard) solution
An aqueous solution of 4β,25-(OH) ₂ D ₃ -d ₄ was prepared at the following concentrations:

(3) Sample pretreatment
(3-1) 200 μL of vitamin D-free serum, which had been adjusted to a concentration of 50 pg/mL of 4β,25-(OH) ₂ D ₃ , and 30 μL of IS were added to a microtube and stirred to prepare a sample.
(3-2) The sample was applied to the antibody column, the lid was closed, and the column was gently mixed.
(3-3) The mixture was mixed and rotated at room temperature for 1 hour using a rotator to allow the antigen-antibody reaction.
(3-4) The outlet of the antibody column was opened, and the column was centrifuged at 550 g for 2 minutes to elute the antibody, and the non-adsorbed fraction was collected.
(3-5) 500 μL of WASHSOL included in the antibody column kit was added, and the mixture was centrifuged at 550 g for 2 minutes twice, and the eluate was discarded.
(3-6) 250 μL of ELUREAG included in the antibody column kit was added, and the column was centrifuged at 550 g for 2 minutes for elution, and the adsorbed fraction was collected.
(3-7) The non-adsorbed fraction was applied to an SLE column, left to stand for 10 min, and then eluted with 600 μL of ethyl acetate/hexane (50/50, v/v). This procedure was repeated three times, and the eluates were combined and concentrated using a centrifugal evaporator.
(3-8) The adsorbed fraction was concentrated directly using a centrifugal evaporator.

(4) Derivatization The adsorbed fraction and the non-adsorbed fraction were each subjected to a derivatization treatment as follows.
(4-1) The derivatization reagent was heated at 80°C for 15 minutes, and then 100 µL was added to the concentrated and dried sample (see Figure 2H).
(4-2) After standing at room temperature for 15 minutes, the mixture was concentrated using a centrifugal evaporator.
(4-3) Dissolve in 50 μL of reconstitution solution (50% acetonitrile aqueous solution).

(5) LC Analysis Conditions After the derivatization treatment, liquid chromatography treatment was carried out under the following conditions.
Equipment: Agilent 1290 Infinity liquid chromatography system
Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 75 mm
Elution conditions: Flow rate 0.4 mL/min. Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile. Detailed elution conditions were as shown in Table 13 below.

(6) MS/MS Analysis Conditions After the liquid chromatography treatment, mass spectrometry was performed under the following conditions.
Apparatus: AB SCIEX QTRAP4500 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode
The SRM parameters were as shown in Table 14 below.

(7) Analysis Results FIG. 4A shows the SRM chromatogram of the antibody column non-adsorbed fraction.
FIG. 4B shows an SRM chromatogram of the antibody column adsorption fraction.
These results confirmed that 4β,25-(OH) _2D3 exists only in the fraction not adsorbed to the antibody column, and that by subjecting the fraction not adsorbed to the antibody column to _{mass spectrometry} , 4β,25-(OH) _2D3 can be separated from other components and detected and/or quantified.

6-5. Example 5: LC-MS/MS analysis _{of 4β,25-(OH)2D3 in} pooled serum

(1) Standard materials: 4β,25-(OH) _{2D3 (} vitamin D compound 54 produced in Example 1 above), 1α,25-(OH) _{2D3 (} SIGMA)
Stable isotope-labeled compounds: 4β,25-(OH) _2D3 - _{d4 (} vitamin D compound 52 produced in Example 1 above) _, 1α, _25- (OH ₎ ^2D3-13C3 (SIGMA)
Vitamin D-free serum: MSG1000 (Golden West Biologicals)
Antibody column: 1,25-(OH) _2- Vitamin _D3 / _D2 Immuno Tube™ Extraction Kit (Immunodiagnostik)
SLE column: Isolute SLE+ column (Biotage)
Derivatization reagent (DAP-PA): JeoQuant Kit for LC-MS/MS Analysis of Vitamin D Metabolites (JEOL Ltd.)

(2) Calibrators _{Calibrators were prepared by adding 4β,25-(OH)2D3 to} vitamin D-free serum at the following concentrations:

(3) IS (internal standard) solution
An aqueous solution of 4β,25-(OH) ₂ D ₃ -d ₄ was prepared at the following concentrations:

(4) Sample pretreatment
(4-1) 200 μL of calibrator or pooled serum, 180 μL of IS, and 20 μL of additive were added to a microtube and stirred to prepare a sample. 10 μL each of 1α,25-(OH) ₂ D ₃ aqueous solution (1 ng/mL) and 1α,25-(OH) ₂ D ₃ - ¹³ C ₃ aqueous solution (1 ng/mL) were added to the calibrator as additives. 10 μL each of 1α,25-(OH) ₂ D ₃ - ¹³ C ₃ aqueous solution (1 ng/mL) and water were added to the pooled serum.
(4-2) The sample was applied to the antibody column, the lid was closed, and the column was gently mixed.
(4-3) The mixture was mixed and rotated at room temperature for 1 hour using a rotator to allow the antigen-antibody reaction.
(4-4) The outlet of the antibody column was opened, and the column was centrifuged at 550 g for 2 minutes to elute the antibody, and the non-adsorbed fraction was collected.
(4-5) 500 μL of WASHSOL included in the antibody column kit was added, and the mixture was centrifuged at 550 g for 2 minutes twice, and the eluate was discarded.
(4-6) 250 μL of ELUREAG included in the antibody column kit was added, and the mixture was centrifuged at 550 g for 2 minutes for elution, and the adsorbed fraction was collected.
(4-7) The non-adsorbed fraction was applied to an SLE column, left to stand for 10 min, and then eluted with 600 μL of ethyl acetate/hexane (50/50, v/v). This procedure was repeated three times, and the eluates were combined and concentrated using a centrifugal evaporator.
(4-8) The adsorbed fraction was concentrated directly using a centrifugal evaporator.

(5) Derivatization
(5-1) The derivatization reagent was heated at 80°C for 15 minutes, and then 100 μL was added to the concentrated and dried sample.
(5-2) After standing at room temperature for 15 minutes, the mixture was concentrated using a centrifugal evaporator.

(6) LC analysis conditions Equipment: Agilent 1290 Infinity liquid chromatography system
Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 100 mm
Elution conditions: Flow rate 0.3 mL/min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile

(7) MS/MS analysis conditions Equipment: AB SCIEX QTRAP4500 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode
The SRM parameters were as shown in Table 18 below.

(8) Analytical Results FIG. 5A shows the calibration curve for the 4β,25-(OH) ₂ D ₃ standard.
FIG. 5B shows an SRM chromatogram of the antibody column non-adsorbed fraction of pooled serum.
FIG. 5C shows an SRM chromatogram of the antibody column adsorption fraction of pooled serum.
These results confirmed that 4β,25-(OH) _{2D3 and} 1α,25(OH) _2D3 were separated, and _that 4β,25-(OH) _2D3 was recovered in the antibody column non-adsorbed fraction, _{and 1α,25(OH)2D3 was recovered} in the antibody column adsorbed fraction.

(9) Quantitative Value _{of 4β,25-(OH)2D3 in} Pooled Serum

Thus, it was found that 4β,25-(OH) ₂ D ₃ could be separated from 1α,25(OH) ₂ D ₃ , and only 4β,25-(OH) ₂ D ₃ could be detected and/or quantified.

Claims (13)

An alkyne compound represented by the following formula (100):
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
(However, the following alkyne compounds:
(Excluding.) An alkyne compound represented by the following formula (100):
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
wherein at least one of _X21 , _X22 , _X23 , and _X24 is any one of CHY, CDY, ^13CHY , and ^13CDY , where Y is any one of _{NH2, 15NH2 ,} OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and ^a sugar substituent;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.] The alkyne compound according to claim 2, wherein in formula (100), at least one of X ₂₁ , X ₂₂ , X ₂₃ , and X ₂₄ is CHY or CDY. A vitamin D compound represented by the following formula (200):
[In formula (200),
A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound;
X' ₁ is C or ¹³ C;
X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ each independently represent any one selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y' is any one _of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other;
R' is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X'3 is C or ^13C ;
At least one of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N;
P is a _C1 - _C10 alkyl group, which may or may not be substituted with a hydroxy group, or a _C2 - _C10 alkenyl group, which may or may not be substituted with a hydroxy group, and the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group;
The sugar substituent has the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]
(However, the following vitamin D compounds:
(Excluding.) A vitamin D compound represented by the following formula (200):
[In formula (200),
A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound;
X' ₁ is C or ¹³ C;
X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ each independently represent any one selected from CHY', CDY', ¹³ CHY', ¹³ CDY', CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y' is any one _of H, D, _NH2 , ^15NH2 , OH, ^18OH , SH, OR', O(CO)R', _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other;
R' is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X'3 is C or ^13C ;
At least one of X' ₁ , X' ₂₁ , X' ₂₂ , X' ₂₃ , X' ₂₄ and X' ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O or ¹⁵ N, wherein at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is modified with a stable isotope ¹³ C, ¹⁸ O or ¹⁵ N, or at least two of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ are modified with a stable isotope D, or at least one of X' ₂₁ , X' ₂₂ , X' ₂₃ and X' ₂₄ is CDD;
P is a _C1 - _C10 alkyl group, which may or may not be substituted with a hydroxy group, or a _C2 - _C10 alkenyl group, which may or may not be substituted with a hydroxy group, and the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group;
The sugar substituent has the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.] 6. The vitamin D compound of claim 5, wherein, in formula (200), P has any of the following structures:
[In the structure relating to P,
Q, Q', Q'', Q''', and Q'''' each independently represent any of H, CH ₂ , CH ₃ , C ₂ H ₅ , O, OH, and a sugar substituent. An analytical method using the vitamin D compound described in claim 4 or 5. The method of analysis includes analyzing a sample by mass spectrometry;
In the analysis, the vitamin D compound is used as an internal standard.
The analytical method according to claim 7. The analysis method according to claim 8, wherein the mass spectrometry is LC-MS/MS or LC/MS. The analytical method according to claim 7, wherein vitamin D compounds that do not have a hydroxy group at position 1 of the A ring are analyzed separately from vitamin D compounds that have a hydroxy group at position 1 of the A ring. The analysis method according to claim 10, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 2-hydroxyvitamin D compound and a 4-hydroxyvitamin D compound. The analysis method according to claim 10, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 4β,25-dihydroxyvitamin D3 compound and a 4-hydroxyvitamin D compound. The method for producing a vitamin D compound according to claim 4 or 5, wherein an alkyne compound represented by the following formula (100) is used as a material for forming the A ring of the vitamin D compound.
[In formula (100),
A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends,
_X1 is CH or ¹³ CH;
X ₂₁ , X ₂₂ , X ₂₃ and X ₂₄ each independently represent any one selected from CHY, CDY, ¹³ CHY, ¹³ CDY, CO, ¹³ CO, C ¹⁸ O and ¹³ C ¹⁸ O;
Y is any one _{of H, D, NH2, 15NH2 ,} ^OH , ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and a sugar substituent, and when two or more Y's are included in formula (100), they may be the same or different from each other;
wherein at least one of _X21 , _X22 , _X23 , and _X24 is any one of CHY, CDY, ^13CHY , and ^13CDY , where Y is any one of _{NH2, 15NH2 ,} OH, ^18OH , SH, OR, O(CO)R, _OSO3H , _OSO3Na , and ^a sugar substituent;
R is an alkyl group, an alkenyl group , an aryl group , or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl ;
_X3 is C or ^13C ;
At least one of X ₁ , X ₂₁ , X ₂₂ , X ₂₃ , X ₂₄ , and X ₃ is modified with a stable isotope D, ¹³ C, ¹⁸ O, or ¹⁵ N;
The sugar substituent has the structure:
With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of such substituents and is an integer of 3 to 5. W is any of H, OH, CH ₂ OH, COOH, NH ₂ , and NHAc, and may be the same or different from each other.]